Pediatr Blood Cancer 2010;55:1201–1203

BRIEF REPORTTrimethoprim-Induced Immune Hemolytic Anemia in a Pediatric Oncology Patient

Presenting as an Acute Hemolytic Transfusion Reaction

Sweta Gupta, MBBS, MD,1 Cindy L. Piefer, MT, SBB,2 Judy T. Fueger, MT, SBB,2 Susan T. Johnson, MSTM, MT, SBB,3

and Rowena C. Punzalan, MD1,3,*

A 10-year-old male with acute leukemia presented with post-chemotherapy anemia. During red cell transfusion, he developedhemoglobinuria. Transfusion reaction workup was negative. Drug-induced immune hemolytic anemia was suspected because ofpositive direct antiglobulin test, negative eluate, and microsphero-cytes on smear pre- and post-transfusion. Drug studies using theindirect antiglobulin test were strongly positive with trimethoprim

and trimethoprim–sulfamethoxazole but negative with sulfamethox-azole. The patient recovered after discontinuing the drug, with norecurrence in 2 years. Other causes of anemia should be consideredin patients with worse-than-expected anemia after chemotherapy.Furthermore, hemolysis during transfusion is not always a transfu-sion reaction. Pediatr Blood Cancer. 2010;55:1201–1203. © 2010Wiley-Liss, Inc.

Key words: drug-induced immune hemolytic anemia; leukemia; trimethoprim

INTRODUCTION

Drug-induced immune hemolytic anemia (DIIHA) is a rareevent, with an incidence of about 1/1,000,000 individuals [1]. Awide spectrum of drugs has been implicated in this phenomenon.More than 80% of DIIHA in the past 10 years have been due tocephalosporins. There has also been an increasing trend seen withbeta-lactamase inhibitors [2]. Immune hemolytic anemia associatedwith trimethoprim (TMP)–sulfamethoxazole (SMX) is rare [3,4].

Most oncology patients are on TMP/SMX prophylaxis for Pneu-mocystis jirovecii. The most common cause of anemia in pediatriconcology patients is chemotherapy-induced bone marrow suppres-sion. We report a case of a child with leukemia who presented withan acute drop of hemoglobin post-chemotherapy and had signs andsymptoms of hemolysis after red blood cell (RBC) transfusion. Insuch a scenario, the first differential is acute hemolytic transfusionreaction. However, our patient was found to have TMP-inducedimmune hemolytic anemia.

CASE REPORT

A 10-year-old Hispanic male with high-risk acute lymphoblasticleukemia (ALL) was admitted for fever and anemia. One day priorto admission, he had completed induction chemotherapy, receivedintrathecal methotrexate and was discharged home with hemoglobinof 10 g/dl. On the day of admission, he presented with dizziness,light-headedness, and temperature of 38.7◦C. After blood cultureswere drawn, he received a dose of ceftriaxone and was transferred toour institution and started on cefepime. Hemoglobin was 8.5 g/dl onadmission and dropped to 6 g/dl 10 hr later. He received 1 U of RBCswithout incident. After receiving 105 ml of a second unit of RBCs,he was noted to have dark-colored urine but no other symptoms. Anacute hemolytic transfusion reaction was suspected.

Initial laboratory evaluation of transfusion reaction is shownin Table I. On the pre-transfusion sample, antibody detection testusing a polyethylene glycol (PEG) indirect antiglobulin (IAT) testtube method was negative and crossmatch was compatible. Directantiglobulin test (DAT) was strongly positive for both IgG (4+)

and complement, C3 (3+). The antibody identification panel wasnegative at room temperature, 37◦C and IAT phase. Non-specificpositive reactions were detected at 18◦C and enhanced after incuba-tion at 4◦C, consistent with a clinically non-significant cold-reactiveautoantibody; this antibody was not reactive at 37, 30, or at 25◦C.A negative Donath–Landsteiner test ruled out paroxysmal coldhemoglobinuria. Prednisone (2 mg/kg/day) was started until the lab-oratory evaluation could be completed.

Labs done at the previous hospital showed heme-positive urine,indicating that the onset of hemolysis may have been before thistransfusion. Pre- and post-transfusion blood smears revealed micro-spherocytes. Lactic acid dehydrogenase (LDH) and unconjugatedbilirubin were also increased pre-transfusion (Table II). These find-ings, along with the strongly positive DAT and negative eluate onboth the pre- and post-transfusion samples, raised the possibility ofDIIHA.

A detailed drug history included several possible associateddrugs. His induction chemotherapy course was complicated bycoagulase-negative Staphylococcus aureus and E. coli sepsis, forwhich he received cefepime and vancomycin for 10 days (stopped 2days before admission), and tobramycin and clindamycin for 2 days(stopped 10 days before admission). He was on acyclovir for 10days (stopped 2 days before admission) for Herpes labialis. He hadreceived 3 days of TMP/SMX for prophylaxis against P. jirovecii7 days before admission; he also received a dose 3 hr prior to the

1Department of Pediatrics (Hematology/Oncology), Medical College ofWisconsin, Children’s Hospital of Wisconsin, Milwaukee, Wisconsin;2Immunohematology Reference Laboratory, Blood Center of Wiscon-sin, Milwaukee, Wisconsin; 3Blood Center of Wisconsin, Milwaukee,Wisconsin

Conflict of interest: nothing to report

*Correspondence to: Rowena C. Punzalan, Department of Pedi-atrics (Hematology/Oncology), Medical College of Wisconsin,MFRC, 8701 W.Watertown Plank Rd, Milwaukee, WI 53226.E-mail: rowena.punzalan@bcw.edu

Received 3 March 2010; Accepted 22 April 2010

© 2010 Wiley-Liss, Inc.DOI 10.1002/pbc.22648Published online 29 June 2010 in Wiley Online Library(wileyonlinelibrary.com).

1202 Gupta et al.

TABLE I. Initial Laboratory Evaluation of Transfusion Reaction

Pre-transfusion Post-transfusion

Urine Heme positivea Heme positive (urine RBC = 0, urine blood = large)Plasma Yellow Red-tingedBlood smear Microspherocytes MicrospherocytesAntibody screen (IAT enhanced with PEG) Negative NegativeDAT

Polyspecific Positive (4+) Positive (4+)Anti-IgG Positive (4+) Positive (4+)Anti-C3 Positive (3+) Positive (3+)Control Negative Negative

Eluate Negative with all cells Negative with all cellsSample drawn at 37◦C ND Insignificant cold-reactive autoantibody reactive at 18 and 4◦CIAT crossmatch Compatible Compatible

DAT, direct antiglobulin test [8]; IAT, indirect antiglobulin test [8]; PEG, polyethylene glycol; ND, not done; LDH, lactic acid dehydrogenase.aOutside hospital results, details of urine RBC unknown; reagents for DAT: polyspecific (rabbit) antihuman globulin (AHG) (Immucor, Inc.,Norcross, GA), monospecific anti-IgG (Immucor, Inc., Norcross, GA), anti-C3d (Ortho Clinical-Diagnostics, Raritan, NJ) and anti-C3b, C3d(Immucor, Inc., Norcross, GA) and saline control.

TABLE II. Laboratory Markers of Hemolysis

Pre-transfusion Post-transfusion After TMP/SMX discontinued

Hematocrit (%) 27.7 21 33.5Blood smear Microspherocytes present Microspherocytes present –LDH (IU/L, normal 370–840) 800 3,320 764Unconjugated bilirubin (mg/dl, normal 0–1.1) 0.9 5.8 0.7

transfusion. He was also on stress doses of hydrocortisone for 10days, which had been stopped 2 days before admission.

Drug studies were performed to look for the presence of drug-dependent antibodies [5]. These included tests done in the presenceof drug (including ceftriaxone, cefepime, vancomycin, tobramycin,clindamycin, and TMP/SMX) and with drug-treated RBCs incu-bated at room temperature and 37◦C and at IAT phase. IAT wasstrongly positive in the presence of TMP/SMX and TMP only butwas negative with SMX only. Additionally drug-dependent RBCantibodies in presence of TMP, but not SMX, were detected byflow cytometry. For this procedure, normal group O RBC’s wereincubated with test serum in the presence and absence of drug andwere washed twice. [RBC-associated immunoglobulins were thendetected by flow cytometry using fluorescein isothiocyanate-taggedantihuman IgG (Fcg-specific) and phycoerythrin-tagged antihumanIgM (Fcm-specific).] These findings were diagnostic of TMP-induced immune hemolytic anemia.

After discontinuation of TMP/SMX and two more RBC trans-fusions, the patient’s hematologic parameters returned to baseline(Table II). Glucose-6-phosphate dehydrogenase (G6PD) level was7.2 U/g Hgb (normal range 7–18 U/g Hgb) on a post-transfusionsample; on repeat, while the patient had been untransfused for >6months, it was normal at 8.3 U/g Hgb with a reticulocyte count of1.9%. The patient was discharged without adverse sequelae, is onpentamidine for Pneumocystis prophylaxis and has had no recur-rence of hemolysis.

DISCUSSION

Our patient had clinical features of immune hemolysis, negativeeluate, positive DAT, and RBC antibodies detected in plasma in the

presence of TMP only, supporting the diagnosis of TMP-inducedimmune hemolytic anemia.

In a patient receiving chemotherapy, the most commonreason for anemia is chemotherapy-induced bone marrow sup-pression. However, when there is an acute drop of hemoglobinimmediately post-chemotherapy, as in our patient, other reasonsfor anemia should be sought. These include bleeding, delayedhemolytic transfusion reaction (DHTR), autoimmune hemolysis,chemotherapy-induced hemolysis, and G6PD deficiency.

The pre-transfusion heme-positive urine in our patient andstrongly positive DAT could have been due either to DHTR to aprevious blood transfusion or drug-induced hemolysis. DHTR wasless likely due the strength of the positive DAT (4+). Addition-ally, the eluate was negative both pre- and post-transfusion. Thispattern of serological reactions is characteristic of drug-inducedimmune-mediated hemolysis. The incidental finding of a clinicallynon-significant cold agglutinin in our patient, can also be seen innormal individuals. Chemotherapeutic agents can cause DIIHA butour patient did not receive any of the agents known to be associatedwith this phenomenon [6]. Studies in the presence of other antibi-otics the patient had been on, including cephalosporins, did not yielda drug-dependent antibody. The positive drug-dependent antibodyin the presence of TMP and not SMX and the temporal association ofthe TMP/SMX dose with hemolysis in this patient supports our con-clusion that this was a TMP-induced immune-mediated hemolysis.The patient continues to take all his medications (except TMP/SMX,which was substituted with pentamidine) and chemotherapy withoutany issues.

DIIHA is identified by clinical evidence of hemolysis associatedwith drug therapy and confirmed by serologic testing. The DATclassically shows strong positive reactivity with anti-IgG, but, it is

Pediatr Blood Cancer DOI 10.1002/pbc

Trimethoprim-Induced Immune Hemolytic Anemia 1203

not unusual to detect C3 on the RBCs of individuals with DIIHA.Petz and Garratty [1] report that 86% of cases of cefotetan-inducedimmune hemolytic anemia have C3 coating on their RBCs. Occa-sionally only C3 may be detected [7].The reaction ranges fromstrongly positive (3–4+, if IgG binding is present) to weakly pos-itive (rarer, due to complement binding) [8]. Weaker positive DATcan also be seen when the patient has been off the associated drugfor some time. DIIHA should be suspected when there is positiveDAT and an with eluate that is non-reactive or with disproportion-ately weaker reactions than the DAT against an RBC panel [7]. Ifinitial studies with native drug are negative then metabolites of thedrug (plasma and/or urine) should be tested [9].

The nature of the serological reactions in our case was consistentwith a drug-dependent antibody that reacts only in the presence ofdrug in its soluble form, causing hemolysis. This drug did not bindcovalently to the RBCs and so could be washed off.

The SMX component of TMP/SMX is known to induce hemoly-sis in patients with G6PD deficiency by inflicting oxidative damageto RBCs leading to destruction. Hemolysis typically occurs 24–72 hr after ingestion, with resolution within 4–7 days [10]. Sinceour patient had been on TMP/SMX for several weeks, had normalG6PD levels and had evidence of immune-mediated hemolysis withTMP, G6PD deficiency as the cause of hemolysis was very unlikely.

We found only two other cases in the literature describingTMP/SMX-induced immune hemolytic anemia. The first case wasof a 10-year-old girl who developed severe hemolytic anemia (pos-itive DAT, elevated LDH, and hemoglobinuria) after receivingTMP/SMX. There was RBC agglutination after incubation withpatient’s serum and TMP/SMX. No testing was done with TMPonly or SMX only [3].The second case was of a 44-year-old womanwho developed DAT-positive hemolytic anemia and renal failureafter TMP/SMX. RBC antibodies were demonstrated in the patient’splasma to both TMP and SMX [4].

The treatment for DIIHA is discontinuation of the drug and sup-portive care for hemolysis. There is generally no role for steroids.After discontinuation of TMP/SMX and more blood transfusions,our patient has had no further evidence of hemolysis.

This case illustrates that although bone marrow suppression isthe most common cause of anemia in oncology patients, other causes

should be considered when there is an acute drop of hemoglobin,including hemolysis. Also, hemolysis during transfusion may notalways be from an acute transfusion reaction. TMP/SMX cannotonly cause hemolysis in association with G6PD deficiency but mayalso be associated with immune-mediated hemolysis.

ACKNOWLEDGMENTS

We are grateful to Brian Curtis, Kathleen E. Puca, MD, NatashaM. Leon, and Krista Bowens of the Blood Center of Wisconsin fortheir contribution to the workup of this patient.

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Pediatr Blood Cancer DOI 10.1002/pbc