Visual inspection of indeterminate crossmatch results prompted polyethylene glycol-enhanced antibody identification in a patient with poor hemoglobin response despite compatible transfusions: a case report

Introduction

Except for naturally occurring anti-A and anti-B, all red cell alloantibodies are categorized as unexpected antibodies. Pregnancy (1), allogeneic blood transfusion (2), transplantation (3), or exposure to immunogenic chemicals can all trigger alloimmunization, which can result in the production of these antibodies. Low-frequency antibodies, low-affinity antibodies (below the detection limit), and missed detection because of a dose effect are the primary causes of ineffective red blood cell (RBC) transfusion. The low detection rate of such alloantibodies poses a significant clinical challenge, as it may lead to acute or delayed hemolytic transfusion reactions (DHTR) (4,5). The International Society of Blood Transfusion (ISBT) and International Haemovigilance Network define DHTR as signs or symptoms of hemolysis occurring from 24 hours to 28 days after transfusion, including unexplained hemoglobin (Hb) drop, dark urine, fever, chills, or laboratory evidence of hemolysis (6). During a DHTR episode, at least one RBC alloantibody is typically found. However, these antibodies might not have been detectable in pre-transfusion crossmatch assays due to their titers falling below the assay’s detection limit.

This case report describes a patient with leukemia. The patient received multiple conventionally crossmatched RBCs transfusions within a short period, but Hb recovery was suboptimal, and abnormal hemolysis markers were observed. Using a polyethylene glycol (PEG)-enhanced tube method followed by column agglutination testing, serologic crossmatching demonstrated incompatibility. After excluding the interference of a positive donor direct antiglobulin test (DAT) on the crossmatch result, the patient’s DAT demonstrated immunoglobulin G (IgG) coating on their RBCs. The antibody eluted from the patient’s RBCs using thermal elution and identified by PEG-enhanced testing was confirmed as anti-E antibody.

This case highlights three key aspects. First, even if routine serologic tests are negative, alloimmunization remains possible. A high level of suspicion should be maintained when there are aberrant hemolysis indicators and an inexplicable weak Hb response. Second, it emphasizes the potential value of visual inspection when automated results conflict with manual interpretation. Third, it demonstrates the use of population-based antigen frequency data as a framework for assessing exposure risk when donor typing is not feasible. We present this article in accordance with the CARE reporting checklist (available at https://aob.amegroups.com/article/view/10.21037/aob-2025-1-57/rc).

Case presentation

A 68-year-old Han Chinese man, blood group A Rh positive and with a negative unexpected antibody screen, presented with a history of chronic myelomonocytic leukemia 7 months earlier and with a history of transfusions and chemotherapy, presented to our hospital for further evaluation and treatment due to severe anemia (Hb: 53 g/L). The patient received three blood transfusions in the first 4 days. After these three transfusions, the patient’s Hb level transiently increased from 53 to 75 g/L. Nine days after the third transfusion, the Hb level progressively declined to 53 g/L, prompting a repeat request for blood products. Over the subsequent 19 days, the patient received a total of 16 units of type A RhD-positive RBCs across nine transfusion episodes. During these 9 transfusions, the Hb level showed an increase after transfusion followed by a decrease (Figure 1). All crossmatches were performed using the indirect antiglobulin test by column agglutination technique. The crossmatching results are shown in Figure 2A.

Figure 1 The levels of Hb, LDH, and TBIL following blood transfusion (the first transfusion was designated as day 1). The red arrows indicate the transfusion of random A RhD-positive RBCs. The blue arrows indicate the transfusion of RBCs with the Rh phenotype R1R1. Hb, hemoglobin; LDH, lactate dehydrogenase; RBCs, red blood cells; TBIL, total bilirubin.

Figure 2 Crossmatch results of the first 13 tests during hospitalization (all compatible, IH-1000). (A) Serial crossmatching results of the patient. (B) Crossmatching result on the 33rd day after the first transfusion. The automated reader reported a compatible result. The enlarged view of the major crossmatch (red frame) shows red blood cells settled at the bottom of the gel column, with a central depression giving a crescent-shaped appearance. PEG-IAT, polyethylene glycol-enhanced indirect antiglobulin.

An additional transfusion was requested on day 33 after the initial transfusion due to a drop in Hb to 52 g/L. Major crossmatch compatibility was recorded by the BIO-RAD IH-1000 automatic blood grouping analyzer; However, visual inspection revealed that the RBCs had settled at the bottom of the gel column, but the surface was irregular with a central depression, forming a crescent-shaped pattern. The result was therefore interpreted as indeterminate, with a ± agglutination strength (Figure 2B). Due to the indeterminate gel column result (crescent-shaped aggregation), crossmatching was performed using the polyethylene glycol-enhanced indirect antiglobulin test (PEG-IAT) technique. The reagent negative control showed no agglutination, while the major crossmatch demonstrated incompatibility, with an agglutination strength of 1+ (Figure 3A). DAT of the crossmatch-incompatible donor units yielded negative results, ruling out a positive donor DAT as the cause of the incompatibility. In contrast, the patient demonstrated a positive DAT with IgG specificity (Figure 3B).

Figure 3 Serologic investigation of the 13th crossmatch incompatibility. (A) Antibody screening (plasma volume: 50 µL) (marked as 1, 2, 3) and major-side PEG-IAT crossmatching. (B) DAT typing results (presented as a table). The patient’s red blood cells demonstrated a positive DAT with IgG specificity. DAT, direct antiglobulin test; IgG, immunoglobulin G; PEG-IAT, polyethylene glycol-enhanced indirect antiglobulin.

A retrospective review of the laboratory results during hospitalization revealed that lactate dehydrogenase (LDH) peaked on day 3 post-transfusion and then began to decline, whereas total bilirubin (TBIL) levels remained persistently above the normal range and fluctuated, increasing and decreasing repeatedly after day 23 (Figure 1).

We performed repeat unexpected antibody screening on post-transfusion samples using multiple methods. Given the weak agglutination observed in crossmatching on the BIO-RAD IH-1000 system, antibody screening was repeated using the manual antiglobulin column agglutination technique with an increased plasma volume of 50 µL instead of the standard 25 µL; the result remained negative (Figure 3A). Given the patient’s positive DAT with IgG specificity, a thermal elution was performed on a post‑transfusion blood sample, and the eluate was tested by antibody screening. Cells 1 and 3 were negative, while cell 2 showed surface protrusions, interpreted as a suspicious positive reaction with an agglutination strength of ± (Figure 4A). Antibody screening of the patient’s plasma using the PEG‑enhanced column agglutination method showed an agglutination strength of 1+ with cell 2 and ± with cell 3 (Figure 4B). The antigen profile of the cells in the antibody screening panel is shown in Figure 4C.

Figure 4 Enhanced detection and identification of unexpected antibodies by PEG-IAT. (A) Antibody screening of the patient’s eluate showed a suspicious positive reaction (±) with cell 2. (B) Antibody screening of the patient’s plasma using PEG-IAT showed an agglutination strength of 1+ with cell 2 and ± with cell 3. (C) Antigen profile of the antibody screening panel cells. (D) Plasma antibody identification (PEG-IAT). The last column (labeled “Ctrl”) is the reagent negative control. (E) Reaction pattern of the antibody identification panel. Red rectangles indicate positive reactions. PEG-IAT, polyethylene glycol-enhanced indirect antiglobulin.

Based on the higher reactivity observed with the PEG-enhanced technique in this case, this method was subsequently used for antibody identification. PEG-enhanced antibody identification was performed using the patient’s plasma against panel cells 1–12 from the Dutch Sanquin antibody identification panel. Agglutination strengths of 1+ were observed with cells 3 and 11, and 2+ with cells 6 and 12; all other panel cells showed negative reactions (Figure 4D). The reaction mode of the antibody identification panel is shown in Figure 4E. Based on the negative exclusion method (a negative reaction with a given RBC indicates the absence of corresponding antibodies in the test sample) and the reaction pattern of the patient’s plasma, the antibody was identified as anti‑E (Figure 4E). Later Rh phenotyping showed that the patient’s Rh phenotype was R1R1 (CCDee), which was consistent with the antibody identification results. All of these tests were completed on the same day that crossmatch incompatibility was observed during repeat crossmatching on day 33 after the first transfusion.

Upon identification of an anti-E antibody, an antigen-matched transfusion strategy was implemented. The patient received two transfusions of E antigen-negative, R1R1 phenotype, crossmatch-compatible RBC units (2 units each) on days 34 and 36. After transfusion, Hb gradually increased, and both LDH and TBIL levels declined. The patient was later discharged. All procedures performed in this study were in accordance with the ethical standards of the institutional and/or national research committee(s) and with the Declaration of Helsinki and its subsequent amendments. Written informed consent was obtained from the patient for the publication of this case report and accompanying images. A copy of the written consent is available for review by the editorial office of this journal.

Discussion

This report described a case of suspected DHTR in a patient with a hematologic malignancy who had received multiple transfusions of crossmatch-compatible RBCs. Upon admission, the patient tested negative for unexpected antibodies. After multiple transfusions, the Hb level showed no significant increase. Although the BIO-RAD IH-1000 system reported crossmatch compatibility, visual inspection revealed an indeterminate result (crescent-shaped aggregation). These concerns were further strengthened by elevated hemolysis indicators such as LDH and bilirubin. This biochemical pattern was consistent with extravascular hemolysis. Subsequent testing via PEG-IAT revealed crossmatch incompatibility. Further investigation using heat elution combined with PEG-IAT confirmed an anti-E alloantibody as the causative agent. A DHTR was strongly suspected. To support the diagnosis of DHTR, we systematically considered and ruled out other possible causes for the poor recovery of the patient’s Hb levels. Firstly, during the observation period, there was no evidence of gastrointestinal bleeding, surgical intervention, or hemodynamic instability, making the possibility of acute blood loss relatively low. Secondly, the pattern of Hb increase followed by progressive decrease after blood transfusion more strongly supported an immune-mediated process rather than bone marrow suppression. Thirdly, since the patient did not use any known drugs that could cause immune hemolytic anemia, drug-induced hemolysis could be ruled out. The insufficient increase in Hb levels after blood transfusion, the persistent elevation of hemolysis markers (LDH and TBIL), the positive IgG-specific DAT, and the subsequent identification of anti-E alloantibodies through PEG-IAT all point more towards the diagnosis of DHTR. We investigated the reasons underlying the repeatedly compatible crossmatch results. Two principal hypotheses were considered to account for the initial serologic compatibility: first, the transfused donor units were E antigen-negative; second, the patient had not yet developed anti-E alloantibodies. In the context of multiple transfusions, inadequate prophylactic Rh (E) antigen matching can precipitate alloimmunization upon exposure to E-positive RBCs.

This retrospective case analysis has several limitations. First, although the residual segments from the 21.5 transfused RBC units remained within their storage window, comprehensive retrospective E antigen typing was not performed. At the time of the acute clinical event, resources were prioritized toward antibody identification and the provision of crossmatch-compatible blood. Therefore, the attribution of hemolysis to antiE remains inferential rather than definitive. The frequency of the E antigen in the Chinese population ranges from approximately 39.05% to 50.8% (7). Applying this frequency to the previous 12 transfusions administered prior to Rh antigen-matched selection, it is statistically probable that 4–6 of these transfusions were E antigen-positive, this represents a probabilitybased inference rather than definitive proof. Second, antibody titration was not performed; consequently, the conclusion that anti-E existed below the detection threshold of routine methods is based on indirect evidence. Third, heat elution was chosen for IgG type antibody identification in this case. For Rh antibodies such as anti-E, ether or acid elution is generally considered more sensitive (8). However, at the time of testing, ether was not available in the laboratory, and clinical urgency required rapid antibody identification to guide ongoing transfusion management. Despite this limitation, heat elution successfully yielded detectable anti-E from the patient’s RBCs. Fourth, the investigation of the patient’s poor Hb response was delayed. Although suboptimal Hb increments and persistently elevated LDH and TBIL levels were observed as early as after the fourth transfusion, the workup for ineffective transfusion was not initiated until after the 13th transfusion. Fifth, the diagnosis of suspected DHTR was made without prospective documentation of all classic clinical symptoms (e.g., dark urine, fever, chills), although laboratory evidence of hemolysis met ISBT diagnostic criteria (6). Finally, as a single case report, the generalizability of our observations is limited. These limitations should be considered when interpreting the findings.

The detection of anti-E by enhanced serologic methods in patients with negative routine screening has been reported in previous studies (9,10). Novaretti et al. compared tube PEG-IAT with gel column agglutination for antibody identification. They found that the gel test detected 196 antibodies missed by PEG-IAT, while PEG-IAT detected only 2 antibodies missed by gel (11). Conversely, a Japanese study tested different methods for detecting IgG alloantibodies. It reported that microcolumn agglutination techniques (Ortho BioVue and DiaMed MTS) had 88.2% sensitivity compared to tube PEG-IAT as the reference standard (100%) (12). Our case is similar to these reports because PEG-IAT also found an antibody that routine testing missed. But there are a few important differences. (I) This case illustrates that when unexplained poor Hb response and hemolysis markers are present, a high index of suspicion for alloimmunization should be maintained—even in the absence of positive serologic results. (II) The crescent-shaped aggregation of RBCs at the bottom of the gel column was not a positive result. However, it served as an important visual clue that prompted further testing. This finding could easily be overlooked. Other laboratories may use it as an early warning sign of possible alloimmunization. (III) A stepwise approach was adopted. Routine gel screening was performed first (negative result). An indeterminate gel morphology was then observed. Next, PEG-IAT was used for confirmation. Finally, elution studies were performed. This approach provides a practical pathway for investigating suspected DHTR when initial results are negative or equivocal; More broadly, it highlights the importance of maintaining a high index of suspicion for alloimmunization when unexplained poor Hb response and hemolysis markers are present, even in the absence of positive serologic results. (IV) Using population-based E antigen frequency data, we demonstrated a statistical framework for estimating the likelihood of E antigen exposure when retrospective donor typing is not feasible, offering a quantitative approach to risk assessment in similar clinical scenarios.

The BIO-RAD IH-1000 automated reader and the visual inspection gave different results in this case. This shows an important problem with current automation. Automated systems are fast and standard, but they may miss small changes in shape that can point to a clinically significant immune response. The crescent-shaped aggregation in this case was the key clue. It was not a positive result, but it prompted further investigation. If the lab had used only the automated reading, this finding would have been missed.

This is important because more and more labs are using fully automated systems. Our case shows that visual inspection is still a key safety step. This is especially true when clinical signs—like poor Hb response or high hemolysis markers—point to an immune problem, even if the automated result says “compatible”. Labs should check automated results by eye when a transfusion reaction is suspected.

Clinical laboratories must be aware of the inherent limitations of serologic compatibility testing. The 24-hour post-transfusion Hb increment serves as a critical efficacy indicator of transfusion efficacy. When this increment falls below projected levels, a diagnostic investigation should be promptly initiated, rather than resorting to escalated transfusion volumes. For patients suspected of DHTR—typically characterized by multiple transfusions, a positive DAT, negative routine antibody screening, and no evidence of acute hemorrhage—the following actions are strongly recommended: (I) optimize antibody detection protocols; (II) initiate enhanced testing algorithms. This includes supplementing routine screening with high-sensitivity methods such as PEG-IAT in gel techniques, enzyme techniques, or using a more sensitive commercial enhancement reagent card. However, PEG-IAT has recognized limitations: (I) it is a manual technique that requires experienced personnel; (II) compared to automated methods, it is more labor-intensive and time-consuming; (III) reduced sensitivity for detecting some antibodies, notably anti-Jk^b; (IV) risk of PEG-induced nonspecific protein agglutination (13,14). Consequently, rigorous RBC washing and appropriate control inclusion are mandatory.

The risk of RBC alloimmunization in patients with hematologic malignancies is heterogeneous and depends on the specific diagnosis and treatment status (15). A large study by Evers et al. showed that patients with acute leukemia on intensive chemotherapy have a much lower risk of alloimmunization [relative risk (RR) 0.36]. Patients with mature lymphomas also have lower risk (RR 0.30). Risk stays lower for up to 6 months after stem cell transplant (RR 0.34, 95% confidence interval: 0.16–0.74). But some blood diseases have higher risk. Evers et al. found that myelodysplastic syndromes (MDS) patients had an 11.2% alloimmunization rate (16). Leisch et al. studied 184 myeloid cancer patients treated with azacitidine. Even with extended matching (ABO, RhD, RhCcEe and K), 20 patients (11%) made alloantibodies. The most common antibody was anti-E. Patients who made antibodies got more RBC units and transfusions over a longer time (17). These findings show that alloimmunization risk in blood cancer patients is complex. It depends on the disease, treatment, and number of transfusions. In our case, the patient made anti-E despite having blood cancer and possible immune suppression. This case illustrates that in the presence of unexplained poor Hb response and hemolysis markers, a high index of suspicion for alloimmunization should be maintained—even when serologic results are negative. For patients who require multiple blood transfusions, specialized transfusion services should implement prophylactic extended antigen matching for crossmatch-compatible RBC units to prevent transfusion-associated alloimmunization (18). At a minimum, this prophylactic matching should encompass Rh (D, C, E, c, e) and K antigens (15,19).

Conclusions

This case shows the important role of visual inspection in transfusion medicine. The automatic system reported that the crossmatch was compatible, and the routine antibody screen was still negative. But visual inspection of the gel showed a result that was not clear—a crescent-shaped pattern. This led to more tests. This visual clue, together with clinical indicators of hemolysis, helped find an anti-E antibody using PEG-IAT. In the presence of unexplained poor Hb response and hemolysis markers, alloimmunization should be suspected despite negative serologic findings. Additionally, this case demonstrates the practicality of using population-based antigen frequency data for risk assessment when donor typing is not possible. These findings reinforce the importance of integrating clinical judgment with serologic findings in transfusion medicine practice.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the CARE reporting checklist. Available at https://aob.amegroups.com/article/view/10.21037/aob-2025-1-57/rc

Peer Review File: Available at https://aob.amegroups.com/article/view/10.21037/aob-2025-1-57/prf

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://aob.amegroups.com/article/view/10.21037/aob-2025-1-57/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All procedures performed in this study were in accordance with the ethical standards of the institutional and/or national research committee(s) and with the Declaration of Helsinki and its subsequent amendments. Written informed consent was obtained from the patient for the publication of this case report and accompanying images. A copy of the written consent is available for review by the editorial office of this journal.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

1. Ko KH, Yoo BH, Kim KM, et al. Frequency of unexpected antibody and consideration during transfusion. Korean J Anesthesiol 2012;62:412-7. [Crossref] [PubMed]
2. Bhuva DK, Vachhani JH. Red cell alloimmunization in repeatedly transfused patients. Asian J Transfus Sci 2017;11:115-20. [Crossref] [PubMed]
3. Monfort M, Honoré P, Gothot A, et al. Simultaneous passenger lymphocyte syndrome and multiple alloimmunization against donor's blood group antigens after liver transplantation. Vox Sang 2015;109:86-90. [Crossref] [PubMed]
4. Hendrickson JE, Fasano RM. Management of hemolytic transfusion reactions. Hematology Am Soc Hematol Educ Program 2021;2021:704-9. [Crossref] [PubMed]
5. Li Q, Xie J, Sun J, et al. Complex delayed blood transfusion reaction: A case report. Medicine (Baltimore) 2024;103:e38467. [Crossref] [PubMed]
6. Definitions T. Hemovigilance definitions by ISBT and IHN. Available online: https://www.tripnet.nl/en/hemovigilance-2/definitions-2/
7. Yu Y, Ma C, Sun X, et al. Frequencies of red blood cell major blood group antigens and phenotypes in the Chinese Han population from Mainland China. Int J Immunogenet 2016;43:226-35. [Crossref] [PubMed]
8. Nathalang O, Sthabunsawasdigarn S, Bejrachandra S, et al. A comparative study of three techniques for eluting red cell antibodies. J Med Assoc Thai 1997;80:S5-8.
9. Panja K, Upreti R. Delayed Hemolytic Transfusion Reaction With Acute Kidney Injury Due to Anti-E Antibody in a 43-Year-Old Woman With Iron-Deficiency Anemia. Cureus 2025;17:e93634. [Crossref] [PubMed]
10. Lemay AS, Faughnan M, Krok E, et al. A curious case of delayed hemolytic transfusion reaction with evanescent antibodies in a patient with hereditary hemorrhagic telangiectasia. Transfusion 2019;59:3570-4. [Crossref] [PubMed]
11. Novaretti MC, Silveira EJ, Filho EC, et al. Comparison of tube and gel techniques for antibody identification. Immunohematology 2000;16:138-41.
12. Sasaki T, Yamada M, Kaneko R, et al. Evaluation of detection sensitivity for red blood cell IgG alloantibodies using various test methods. Japanese Journal of Transfusion Medicine 2003;49:640-5.
13. Robinson MW, Scott DG, Bacon PA, et al. What proteins are present in polyethylene glycol precipitates from rheumatic sera? Ann Rheum Dis 1989;48:496-501. [Crossref] [PubMed]
14. Watanabe K, Takeuchi C, Yokota M, et al. False negative agglutination phenomenon in hyperglobulinemia serum in the polyethyleneglycol indirect anti-globulin test. Journal of the Japan Society of Blood Transfusion 2002;48:342-9.
15. Pattarakosol P, Lorucharoen N, Watanaboonyongcharoen P, et al. Risk factors for red blood cell alloimmunization in patients with hematologic malignancy. Transfus Med 2024;34:499-505. [Crossref] [PubMed]
16. Evers D, Zwaginga JJ, Tijmensen J, et al. Treatments for hematologic malignancies in contrast to those for solid cancers are associated with reduced red cell alloimmunization. Haematologica 2017;102:52-9. [Crossref] [PubMed]
17. Leisch M, Weiss L, Lindlbauer N, et al. Red blood cell alloimmunization in 184 patients with myeloid neoplasms treated with azacitidine - A retrospective single center experience. Leuk Res 2017;59:12-9. [Crossref] [PubMed]
18. Pathak A, Tejwani N, Panda D, et al. Determination of the Rh/Kell phenotypes in donor as well as patients might be significant to provide phenotype-matched blood to cancer patients: A retrospective analysis from a tertiary care oncology center in North India. Asian J Transfus Sci 2023;17:234-8. [Crossref] [PubMed]
19. Wemelsfelder ML, van de Weem RHG, Luken JS, et al. Extensive red blood cell matching considering patient alloimmunization risk. Vox Sang 2024;119:368-76. [Crossref] [PubMed]