Potential serological challenges caused by anti-IH antibody in the crossmatch laboratory.

Sir,

Cold auto-agglutinins constitute the human sera and react optimally at lower temperatures ranging anywhere from 0°C to 4°C.[1] These antibodies are clinically benign, having low titers and demonstrate a very narrow thermal amplitude (TA).[2] Likewise, anti-IH is a complex cold agglutinin having a benign nature and preferentially act at lower temperatures. Its demonstration requires a co-expression of both I and H antigens on the erythrocyte membrane (EM). Further, the reactivity of anti-IH usually depends on the amount of H antigens present on the EM, which makes it react more with O and A2 cells as against A1 and A1 B cells.[2] Seldom, this may present as a clinically significant antibody resulting in an acute hemolytic transfusion event.[3] We describe herein, an instance of a 42-year-old male having aplastic anemia and past history of cerebrovascular accident who was admitted for the management of his anemia at our hospital. He experienced a recent history of blood transfusion almost 10 days back from an outside facility. His hematological investigations revealed a low hematocrit of 19.7% that necessitated a packed red blood cell (PRBC) transfusion. The pretransfusion work up was performed according to our departmental standard operating procedures. His blood grouping by conventional tube technique showed a discrepancy. Whereas, cell grouping suggested A1 Rh (D) positive, his serum showed varying grades of agglutination with A1, B, and O pooled erythrocytes. It is worthwhile noting that his serum showed a higher grade of agglutination especially with the O pooled erythrocytes as against A1 cells, respectively [Table 1]. Repeat serum grouping after incubation at 4°C and 37°C [Table 2] for 30 min each, showed an irregular antibody having a preferential action at 4°C, hence bringing up the suspicion of a cold agglutinin. The irregular antibody did not resolve completely even at 37°C and showed a weaker grade of reaction (w+). This was hence, typed as type IV discrepancy with the presence of an unresolved irregular cold antibody. His direct Coombs test and auto-control both were negative. On performing the antibody screening and identification with the commercially available Diamed Gel cards (Biorad, Switzerland), pan-agglutination was observed. Cold antibodies such as anti-I and anti-H were ruled out since patients' serum showed weaker (w+ and 1+) grades of agglutination with Oh (Bombay) I+ adult cells and Oi (Cord) H+ cells, respectively. The reaction pattern, however, agreed with another cold agglutinin namely, anti-IH. On performing serial dilutions of patients' serum with O (I+ H+) adult erythrocytes at 4°C, 18°C–22°C and 37°C temperatures, resultant titers obtained were 32, 8, and 4, respectively. We also treated patients' serum with dithiothreitol (DTT) that yielded a zero grade in the first tube while, with phosphate buffer saline (PBS) it showed the titer of 16 and 4 both at saline as well as the anti-human globulin (AHG) phase, respectively. Broad TA rather than its titer was inferred to be a critical measure depicting the clinical significance of the underlying antibody. While cross-matching patients' serum with donor erythrocytes, A1 cells showed a reduced grade of reactivity pattern typically ranging from 0 to 1+ as against higher grade of reactivity (4+) with O cells. The patient was eventually transfused with one pint of AHG cross-matched compatible A1 Rh (D) positive PRBC using a blood warmer without any adverse consequence.

Table 1

Patient blood grouping performed by the conventional tube technique

Anti-A	Anti-B	Anti-D	Anti-A1 lectin	Auto control	A1 pooled cells	B pooled cells	O pooled cells	Interpretation
4+	0	4+	3+	0	2+	4+	4+	A1 Rh (D) positive with? irregular antibody

The significance is towards the interpretation of varying grades of agglutination ranging from 0 to 4+

Table 2

Repeat serum grouping at different temperature ranges

Temperature (°C)	A1 pooled cells	B pooled cells	O pooled cells	Interpretation
4	3+	4+	4+	? Irregular cold antibody with a broad TA
37	1+	4+	3+

The significance is towards the interpretation of varying grades of agglutination ranging from 0 to 4+. TA=Thermal amplitude

In general, cold auto-agglutinins are directed against the Ii blood group collection.[2] Therefore, because of the biochemical relationship of ABH and Ii antigens, it is not surprising that ABH–Ii complex specificities (e.g., anti-IH and anti-IA) that require the presence of more than one antigen to react, is widely described in the literature.[4] Just like other cold agglutinins (with narrow TA), anti-IH also does not usually interfere during the pretransfusion testing; in fact these can often be circumvented on avoiding room temperature testing phases. Albeit, sometimes, anti-IH can behave as a clinically significant entity showing a broad TA. In this case, several factors including, its serologic presentation, potency, and specificity helped us clinch the diagnosis bench-side. Even the reactivity pattern in serum typing with A1 pooled erythrocytes (having reduced H antigen expression) appeared weaker when compared to the O pooled cells. Because H antigen is the substrate for A and B antigens, the expression of A and/or B antigens is always reciprocal to its expression. While group O erythrocytes have the maximum expression of H antigen, group A1 B erythrocytes have the least (O>A2>B>A2B>A1>A1B).[3] Mohanan et al. have described a similar instance of a clinically significant anti-IH antibody showing a broad TA.[5] In our example, the anti-IH antibody demonstrated a higher grade of reactivity pattern and broader TA with group O adult erythrocytes as against group A1 adult cells. Further, there was reduced reactivity with group Oi (cord) cells as well as Oh (Bombay) cells, respectively. Reduced reactivity of DTT-treated serum and TA studies pointed toward an IgM characteristic of the antibody. Broader TA was deduced from the fact that the reactivity pattern with group O adult (I+ H+) erythrocytes matched at both room temperature as well as 37°C. At the time of discharge, the patient's hematocrit was 24.7% and his overall condition was clinically stable.

To conclude, we reiterate the fact that a cold agglutinin of anti-IH specificity having a broader TA, although rare, can be potentially catastrophic, and a timely identification of the same can help circumvent any probable adverse event in the recipient.

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Conflicts of interest

There are no conflicts of interest.