Clinical Utility of Flow Cytometry for Detection of Anti-Jk^b IgM in Acute Haemolytic Transfusion Reaction

Abstract

Acute hemolytic transfusion reaction (AHTR) is a rare but life-threatening complication of transfusion. We herein report a case of anti-Jk^b IgM-related AHTR. Two hours after an 80-year-old man with myelodysplastic syndrome received a packed red blood cell (RBC) A⁺/Rh^-/Jk^b+/c^- transfusion, he developed acute respiratory failure and a fever. Although he had tested negative in routine screening tests, the 37°C normal saline test was weakly positive for Jk^b. We confirmed the presence of anti-Jk^b IgM in the patient's serum by flow cytometry. This case demonstrates the potential utility of flow cytometry for IgM detection.

Introduction

Acute haemolytic transfusion reaction (AHTR) due to Kidd system antibodies is a rare life-threatening complication associated with transfusion (1,2). AHTR is caused by immune reactions mediated by preformed antibodies (IgG or IgM) in the recipient that bind donor red blood cell (RBC)s (3,4). In addition to the 37°C normal saline test method for detecting IgM antibodies, flow cytometry (FCM)-based methods have attracted attention as promising tools for antibody detection (5,6). However, there have been no reports describing the utility of FCM in AHTR because of the rarity of the condition.

We herein report a case of AHTR caused by anti-Jk^b IgM, for which an FCM-based method was useful.

Case Repot

One year ago, an 80-year-old man (A⁺/Rh^-/Jk^b-/c^-) had been admitted to our hospital and diagnosed with myelodysplastic syndrome. He had no history of transfusion. Six months after the diagnosis, the patient received A⁺/Rh^-/Jk^b+/c⁺-compatible standard packed RBC units (240 mL of packed RBCs) for the first time because of progressive anemia.

One month later (seven months after the diagnosis), the patient was treated with another standard packed A⁺/Rh^-/Jk^b+/c^- RBC transfusion. A pre-transfusion evaluation showed no apparent abnormalities. Two hours after the second transfusion, however, the patient developed dyspnea (SpO₂ 80% in ambient air), tachycardia, and a fever (39°C). Biochemical data revealed intravascular hemolysis, including elevated lactose dehydrogenase (1,051 IU/L) and undetectable haptoglobin levels. The serum and urine samples were brownish in color. Chest radiography and computed tomography revealed no pulmonary edema or acute respiratory distress syndrome. He was diagnosed with AHTR, and non-invasive positive-pressure ventilation was initiated owing to severe respiratory failure (PaO₂/FiO₂ ratio=120). Direct antibody testing (DAT) was negative for IgG and C3 antibodies. After intensive care, the patient was discharged 10 days following the transfusion, without sequelae.

An evaluation of the preserved plasma sample obtained before transfusion following AHTR by automated column agglutination was positive for anti-c antibodies only. A dithiothreitol dissociation analysis on the day after AHTR showed no apparent IgG or complement levels. However, the 37°C normal saline test showed weak positivity for the anti-Jk^b antibodies.

We then performed FCM-based IgM detection using Jk^b+ and Jk^b- RBCs. In brief, 3% type O and 3% Jk^b+/c- and Jk^b-/c^- RBCs were incubated with the patient's serum at 37°C for 30 minutes. The RBC pellet was resuspended and adjusted to a concentration of 10⁷ cells/mL. The specimens were incubated with fluorescein isothiocyanate (FITC) anti-human IgM (BioLegend, San Diego, USA) and PE anti-human CD235a (BioLegend) for 30 minutes. We then compared the mean fluorescence intensity (MFI) of RBCs using FCM (BD Fortessa, Franklin Lakes, USA).

As shown in Figure, FCM showed a higher MFI in Jk^b+/c- RBCs than in Jk^b-/c- RBCs (donor and patient MFI were -17.4 and 37.8, respectively). The patient was thus diagnosed with anti-Jk^b IgM-related AHTR.

Figure.

Jk^b-IgM detection by flow cytometry. A: Schematic illustration for FCM-based Jk^b-IgM detection. B, C: Histograms showing representative data of CD235a⁺ RBCs.

After the diagnosis, the patient was successfully treated with Jk^b-/c^- RBC transfusions. Thereafter, no hemolytic attacks were observed. Seven days after AHTR, a further analysis using an enzyme method (2 step papain method) at the Japanese Red Cross Society laboratory center showed that he was positive for Jk^b. Two months after the hemolytic attack, the patient tested positive for Jk^b using the column agglutination method (Table). We concluded that the patient had developed AHTR associated with IgM during his primary immune response against Jk^b rather than with IgG.

Table.

Results of Various Methods for Irregular Antibody Detection in the Current Case.

		1 month before transfusion		Following day of transfusion		7 days after transfusion		2 months after transfusion
DAT		-		n.d.		-		n.d.
Column agglutination		-		-		-		+
LISS-IAT		n.d.		n.d.		-		+
PEG-IAT		-		-		+		+
Bromelain (one-step)		-		n.d.		-		+
Papain (two-step)		n.d.		n.d.		+		n.d.
Normal saline (37 degree celsius)		n.d.		+		n.d.		n.d.

Discussion

Our case demonstrates the utility of FCM in detecting anti-Jk^b IgM in patients with AHTR. A literature review showed only a few cases of IgG- and IgM-associated AHTR caused by Kidd-type antigens (2,7). Of these, only one case was implied to have IgM-related hemolysis because of negative results of standard DAT (2); however, the authors could not confirm the presence of serum anti-Kidd IgM in the serum. The rarity of anti-Jk^b ATHR is due to the low antigenicity of Jk^b [calculated at 0.04% per transfusion (8,9)] and the limited circulation period of IgM. However, appropriate evaluation methods are warranted given the lethal clinical course of AHTR.

Despite the severe clinical course, DAT for complement in our case was negative, as observed in previous cases (2). We considered this result to be pseudo-negative, as the serum complement levels might have declined due to severe hemolysis. As FCM was capable of identifying RBC-binding immunoglobulins more sensitively than DAT [limit of 500 immunoglobulins per cell (10)], we attempted an FCM-based evaluation. Recently, the clinical efficacy of FCM has been established, especially for Coombs-negative autoimmune hemolytic anemia and blood typing (10). In the present case, we successfully confirmed IgM-related AHTR using FCM.

Our case is important because it shows that Jk^b can cause AHTR, which can be detected using FCM-based IgM detection. Our case highlights the utility of FCM with respect to both sensitivity and speed, which is required in scenarios demanding an urgent subsequent transfusion. Although further validation studies are required, our case suggests that FCM can also be applied to other irregular antibodies and that an FCM-based analysis is a promising tool for IgM-AHTR detection.

Written consent was obtained from all patients. This case report was approved by the Institutional Ethics Committee (Institution Ise Red Cross Hospital ethical committee).

The authors state that they have no Conflict of Interest (COI).