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\begin{document}

\title{Posttransfusion hyperhemolysis syndrome in beta thalassemia major:
Postulation of various mechanisms}



\author[1]{Ganesh Kasinathan}%
\author[2]{Jameela Sathar}%
\affil[1]{Ampang Hospital}%
\affil[2]{Ampang Hospital.}%


\vspace{-1em}



  
  \date{May 25, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Hyperhemolysis syndrome (HS) should be considered in a multiply
transfused thalassemia patient. HS in this patient was attributed to a
combination of factors including multiple transfusions, presence of
anti-Cw, macrophage hyperactivity, hypersplenism and suppression of
erythropoiesis.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Hyperhemolysis syndrome (HS), a potentially life-threatening condition,
was first described in sickle cell disease but was subsequently seen in
other hematological conditions such as thalassemia, myelofibrosis and
lymphoma\textsuperscript{1}. This syndrome is characterised by the
occurrence of severe anemia with posttransfusion hemoglobin levels being
lower than pretransfusion levels\textsuperscript{2}. Intravascular
destruction of both host and transfused red blood cells occurs as
evident by the presence of positive hemolytic markers such as indirect
hyperbilirubinemia, elevated lactate dehydrogenase (LDH),
hemoglobinuria, severe reticulocytopenia and decreased serum
haptoglobin. HS is categorised into acute and delayed forms. The acute
form occurs within the first 7 days of red cell transfusion meanwhile
the delayed form occurs more than 7 days post red cell
transfusion\textsuperscript{3}. HS rarely recurs. Here, we describe the
possible mechanisms involved in a transfusion dependent beta thalassemia
major patient presenting with acute hyperhemolysis syndrome after red
cell transfusion.

\textbf{Case presentation}

A 45-year-old gentleman of Chinese ethnicity presented to the
thalassemia ward with high grade fever associated with black urine,
generalised bone pain and worsening jaundice 36 hours after being
transfused with 2 units of ABO and Rh matched leukoreduced cross match
compatible red blood cells. He was a known beta thalassemia major, in
which, he underwent regular two-weekly transfusion since childhood.
However, he was occasionally non-compliant to his transfusion schedule
with the latest blood transfusion being 6-weeks prior. He had
extramedullary hematopoiesis in the liver, spleen and paraverterbra with
mild liver hemosiderosis. He was on dual oral iron chelation therapy. He
had a family history of beta thalassemia. He was a non-smoker, a
teetotaler and never consumed traditional drugs. He worked as a lawyer.

Physical examination revealed a thin-built gentleman who was
significantly pallor and jaundiced. He was febrile at
39.5\textsuperscript{0}C with a blood pressure of 140/80 mmHg and
tachycardic at 112 beats/minute. He did not demonstrate any cutaneous
rash. He had a palpable liver of 5 cm and a spleen of 15 cm. Other
systemic examinations were unremarkable.

His pre-transfusion hemoglobin (Hb) of 5.2g/dL decreased further to 4.6
g/dL after 2 units of red cell transfusion. He had a steady state mild
pancytopenia (total white cell count: 3.2 x 10\textsuperscript{9}/L and
platelet: 75 x 10\textsuperscript{9}/L) which was attributed to his
extramedullary haematopoiesis-related-hypersplenism. His hemolytic
markers and peripheral blood film on day 2 of transfusion were
consistent with acute intravascular hemolysis. He also had marked
hyperferritinemia (his baseline serum ferritin: 200-300 ug/L). His blood
and urine cultures were both sterile excluding the possibility of
bacterial contamination. The other laboratory parameters are tabulated
in Table 1.

Further investigations were performed at the blood bank on Day 2 of
transfusion. His ABO and Rh blood grouping on conventional tube
technique was O Rh(D) positive, R1R1 phenotype which were the same
previously. The micro Coombs (Direct antiglobulin test-DAT) using low
ionic strength saline (LISS)/anti-human globulin (AHG) column
agglutination card was 2+ for anti-C3d and negative for anti-IgG. The
antibody screening on patient's serum using ID Diacell Asia I-II-III
(LISS/AHG column agglutination card) at 37\textsuperscript{0}C did not
demonstrate any unexpected antibodies on Day 2. A repeated micro Coombs
test at Day 15 was negative for both anti-IgG and C3d. Furthermore, the
antibody screening showed presence of alloantibody in cell III on Day 15
as shown in Table 2. A 11-cell antibody identification panel (Table 3)
identified anti-C\textsuperscript{w} in the patient's serum. The
patient's RBCs were genotyped using sequence specific primer-polymerase
chain reaction (SSP-PCR) molecular method as RHCE*C/C, RHD*01/01,
RHCE*e/e, JK*A, JK*B, KEL*k, FY*A, GYPA*N, GYPB*s. Hence, the RBC
phenotype was predicted as R1R1, C\textsuperscript{w}-, Jka+b-, Fya+b-,
kk, NN, ss.

A diagnosis of hyperhemolysis syndrome was made. He was treated with a
synergistic combination therapy of intravenous methylprednisolone 8mg/kg
daily and intravenous immunoglobulin (IVIg) at a dose of 0.4g/kg daily
for a 5-day duration. He was also hydrated adequately with isotonic
saline, given subcutaneous erythropoietin-alpha and prophylaxis
anticoagulation with enoxaparin 40mg daily (weight 58kg) to prevent
hemolysis-induced-thrombosis. The Hb decreased to a nadir of 3.5g/dL at
day 10 before increasing to 5.8g/dL at day 15 without any blood
transfusion. He was counselled for splenectomy, in which, he refused for
the fear of surgery. He is currently on two weekly crossmatch compatible
leukoreduced RBC phenotype matched red blood cell transfusion at our
thalassemia daycare center.

\textbf{Table 1}\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
Parameters (normal range in S.I.unit) & Day 2 & Day 6 & Day 10 & Day
15\tabularnewline
\midrule
\endhead
Haemoglobin (13.5-16.0 g/dL) & 4.6 & 4.2 & 3.5 & 5.8\tabularnewline
Reticulocyte count (50-100 x 10\textsuperscript{9}/L) & 15.0 & 14.0 &
12.0 & 28.0\tabularnewline
Lactate dehydrogenase (180-250 U/L) & 688 & 522 & 319 &
265\tabularnewline
Haptoglobin (0.3-2.0 g/L) & 0.0737 & & 0.32 &\tabularnewline
Indirect Bilirubin (3.4-12.0 umol/L) & 135 & 94 & & 23\tabularnewline
Urine haemoglobin & 2+ & & 0 &\tabularnewline
Creatinine (74-107 umol/L) Ferritin (24-336 ug/L) & 58 6700 & & 56 1550
&\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table 1} Haematological and biochemical parameters

\textbf{Table 2}\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
\begin{minipage}[b]{0.22\columnwidth}\raggedright\strut
Incubation phase\strut
\end{minipage} & \begin{minipage}[b]{0.22\columnwidth}\raggedright\strut
IS\strut
\end{minipage} & \begin{minipage}[b]{0.22\columnwidth}\raggedright\strut
LISS/AHG\strut
\end{minipage} & \begin{minipage}[b]{0.22\columnwidth}\raggedright\strut
CCC\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
Cell I\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
Cell II\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
Cell III\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
1+\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
AC\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
NT\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

Cell followed by a Latin number indicates antibody screening cell; IS:
immediate spin; LISS: low ionic strength saline; AHG: anti-human
globulin; CCC: Coombs control cells, AC: auto control, NT: not tested

\textbf{Table 2} Antibody screening test on Day 15 red cell transfusion

\textbf{Table 3}\selectlanguage{english}
\begin{longtable}[]{@{}lllllllllllll@{}}
\toprule
\begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
Cell No\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
Rh-hr C\textsuperscript{w}\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
Kell\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
Duffy\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
Kidd\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
Lewis\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
MNS\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
P\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
Luth.\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
Xg\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
LISS/ AHG\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
Enzyme (papain)\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
CCC\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Cell 1\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
+\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
2+\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
4+\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Cell 2\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
2+\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Cell 3\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
2+\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Cell 4\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
2+\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Cell 5\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
2+\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Cell 6\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
2+\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Cell 7\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
2+\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Cell 8\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
2+\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Cell 9\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
2+\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Cell 10\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
2+\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Cell 11\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
2+\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
AC\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
NT\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

Cell followed by a Latin number indicates antibody screening cell; LISS:
low ionic strength saline; AHG: anti-human globulin; CCC: Coombs control
cells, AC: auto control, NT: not tested

\textbf{Table 3} Antibody identification panel on Day 15 red cell
transfusion

\textbf{Discussion}

We describe an unusual presentation of hyperhemolysis in a beta
thalassemia major patient who presented with high grade fever, bone pain
and malaise. It was interesting to note that Day 2 DAT was negative for
anti-IgG but showed 2+ for anti-C3d. A well performed DAT detects
100-500 molecules of bound IgG per red blood cell. A negative IgG
autoantibody is a common feature in acute form of HS. There are several
reasons which may explain a negative DAT in HS. They are (a)
sensitization of the patient's red cells with a small number of IgG
molecules which is below the threshold for detection by a standard DAT
(b) most standard DAT do not incorporate anti-IgA monoclonal reagent,
thereby, missing out on the possibility of IgA mediated hemolysis (c)
low affinity IgG autoantibodies being eluted from RBC during the washing
process\textsuperscript{4}. A super Coombs test, which incorporates an
enhanced and more sensitive version of the standard DAT may be useful in
cases where the standard DAT is negative.

It is also interesting to note that the negative anti-C3d on Day 15
repeated DAT in our patient could be attributed to the immunosuppressive
corticosteroid therapy given to him.

The pathogenesis of HS is interesting. The mechanisms which may explain
HS in our patient are: (a) macrophage hyperactivation (b) RBC
alloimmunisation (c) complement regulation dysfunction (d) bystander
hemolysis (e) suppression of erythropoiesis and (f) HLA
alloimmunisation\textsuperscript{5}. Macrophages are widely believed to
be in a primed state in hemoglobinopathy syndromes. In response to
allogeneic transfusion, macrophages become hyperactivated and bind to
both host and donor red blood cells resulting in rapid
destruction\textsuperscript{5}. Interestingly, our patient had
significant extramedullary hematopoiesis in the spleen resulting in
hypersplenism. Since the splenic red pulp contains a large number of
macrophages, therefore this group of patients are at considerable risk
of developing HS.

Bystander hemolysis, a term coined by Petz and Garraty, is used to
describe immune hemolysis of erythrocytes in the presence of recipient
alloantibodies directed to antigen negative transfused red blood
cells\textsuperscript{6}. Besides RBC alloantibodies; HLA and plasma
protein antigens from the transfused blood may react with the
recipient's antibodies contributing to complement regulation
dysfunction, thereby leading to bystander hemolysis\textsuperscript{7}.
However, in this patient, we did not perform an HLA antibody screen, and
facilities to measure levels of complement factors, C5b-9 to support our
findings of complement dysfunction were unavailable.

Besides the above, RBC alloimmunization is also an important cause of
hyperhemolysis. A negative antibody screen in our patient at Day 2 of
transfusion is a feature commonly seen in the acute form of HS. Standard
serological studies used may not be potent enough to detect low affinity
alloantibodies against low frequency antigens. Antibody dependent cell
mediated cytotoxicity (ADCC) may explain the mechanism of hemolysis in
recipients with levels of alloantibody below the serological detection
threshold\textsuperscript{8}. Interestingly,
anti-C\textsuperscript{w}was subsequently detected in our patient's
serum on Day 15. This antibody including those of the Kidd or Rh system
are often undetectable on pretransfusion testing but may increase
rapidly in titer following a blood transfusion. Re-exposure of an
antigen negative recipient to a red cell antigen positive transfusion
could result in an anamnestic antibody response, precipitating
hemolysis. Anti-C\textsuperscript{w} is an alloantibody directed against
the C\textsuperscript{w} antigen (Rh 8), which is located on the RHCE
gene. This would explain the initial presence of anti-C3d on Day 2 DAT
in which, the anti-C\textsuperscript{w} causes activation of the
complement. C\textsuperscript{w} is a low incidence antigen, and has
been reported to be 1\% and 2\% in Blacks and Caucasians
respectively\textsuperscript{9}. Anti-C\textsuperscript{w}, usually an
IgG antibody, may occur naturally or after an immune stimulation by
blood transfusion such as seen in this patient. Unlike other Rh
antigens, anti-C\textsuperscript{w} is usually not significant.
Anti-C\textsuperscript{w} associated moderate-to-severe hemolytic
disease of the fetus and newborn (HDFN) have been reported infrequently
in case reports and thus may be of clinical significance in females of
reproductive age\textsuperscript{10}. However, literature on the
significance of this antibody in acute and delayed hemolytic transfusion
reaction is severely lacking.

Severe reticulocytopenia was also an additional feature seen in our
patient. Thalassemia patients are known to have significantly lower
serum levels of erythropoietin at steady state which may trigger
neocytolysis during HS causing further destruction of erythroid
precursors\textsuperscript{11}. A bone marrow analysis would be helpful
to confirm the presence of erythropoiesis suppression. Another mechanism
which could also contribute to reticulocytopenia is increased peripheral
consumption of reticulocytes by hyperactive macrophages in the presence
of preserved bone marrow activity of erythropoiesis.

Management of HS is largely dependent on the severity of anemia and
degree of hemolysis. Blood transfusion should be avoided in the milder
form of HS as they are known to exacerbate hemolysis. Combination
therapy of IVIg and steroids are synergistic in suppressing macrophage
activation and shortening hemolysis through immodulatory
effects\textsuperscript{12}. Low dose IVIg at 0.4g/kg daily for 5 days
and intravenous methylprednisolone 500mg daily for 2 days are
recommended for the treatment of HS\textsuperscript{12}. In severe forms
of HS, red blood cell transfusion may be required but should be given in
the presence of IVIg-steroid cover. Subcutaneous erythropoietin may have
a role in direct stimulation of erythropoieis and preventing
neocytolysis. Furthermore, since macrophage activation is an important
mechanism in HS, anti-CD20 monoclonal antibody such as rituximab
375mg/m\textsuperscript{2} even at a single dose has shown
response\textsuperscript{13}. It is interesting to note that eculizumab,
a C5 convertase inhibitor which inhibits complement activation is
promising in the treatment of HS in sickle cell anemia and may also
confer such benefit in thalassemia\textsuperscript{14}. On another note,
as hemolysis increases the risk of both arterial and venous thrombosis,
it is imperative to ensure adequate anticoagulation prophylaxis is given
during active HS. Splenectomy has a role in HS as it is known to resolve
HS crisis in thalassemia\textsuperscript{15}. However, our patient
declined splenectomy due to personal reasons.

\textbf{Conclusion}

A high index of suspicion of HS allows prompt institution of therapy
resulting in good survival outcome. The occurrence of HS in our patient
is attributed to a constellation of multiple factors namely; chronic
multiple transfusions, presence of anti-C\textsuperscript{w}, macrophage
hyperactivity, extramedullary haematopoiesis-induced-hypersplenism and
suppression of erythropoiesis.

\textbf{Declarations}

Ethical approval: Ethical approval is not required as this is not a
clinical trial.

Consent: Written informed consent was obtained from the patient for
publication of this case report and the accompanying images. A copy of
the written consent is available for review by the Editor-in-Chief of
this journal on request.

Competing interests: The authors declare there are no conflicting
interests.

Funding and sponsorship of this paper: Self-funding and no sponsorship
received.

Author's contribution: G.K. analysed the data, designed the paper and
wrote the first draft of the manuscript. J.S. made critical revisions
and approved the final manuscript.

Guarantor: Ganesh Kasinathan is the guarantor of this manuscript.

Acknowledgements: None

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