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®TRANSFUSIONMEDICINENO. TM 11-4 (TM-328)Transfusion medicineVolume 54Brian Nagao, MDPathology ResidentShan Yuan, MDAssistant Professor of Pathology and Laboratory MedicineAssistant Director, Transfusion Medicine & Blood BankingNumber 4ISSN 1056-5949© ascp May 2011Qun Lu, MDAssistant Professor of Pathology and Laboratory MedicineAssistant Director, Transfusion Medicine & Blood BankingDepartment of Pathology and Laboratory MedicineDavid Geffen School of MedicineUniversity of California at Los AngelesLos Angeles, CaliforniaIf you wish to obtain an MOC self-assessment module (SAM) creditfor this exercise, please log-in to www.ascp.org, go to My Courses, andselect the appropriate course now to take your pre-test.41

LEARNING OBJECTIVEStransfusion medicineTM 11-4© ASCP 2011Upon completion of this exercise, the participant should be able to• recognize immune-mediated hemolysis.• develop a differential diagnosis for immune-mediated hemolysis.• diagnose drug-induced immune hemolytic anemia.• diagnose drug-induced immune thrombocytopenia.• recommend appropriate management for both conditions.Copyright © 2010 2011 by the American Society for Clinical Pathology. All rights reserved. Copyright law allows formultiple copy duplication of the Check Sample exercises and color images (printed or digital) for classroom useand for in-service continuing education by nonprofit groups. Written permission is not required in these casesas long as copies made do not exceed the number of students or participants and copies are not charged for. Allother uses including reproduction, storage in a retrieval system, or transmission in any form or by any means(including electronic, mechanical, photocopying, or recording) require prior written permission of the AmericanSociety for Clinical Pathology, checksample@ascp.org. Check Sample® is a registered trademark of the AmericanSociety for Clinical Pathology.The American Society for Clinical Pathology (ASCP) is accredited by the Accreditation Council for ContinuingMedical Education to provide continuing medical education (CME) for physicians.The ASCP designates this educational activity for 1 AMA PRA Category 1 Credit TM . Physicians should only claim creditcommensurate with the extent of their participation in the activity.These activities meet the American Board of Pathology’s Maintenance of Certification Program Part II Lifelong Learningrequirements.Select ASCP continuing education activities are accepted by California, Florida, and many other states for relicensureof clinical laboratory personnel. ASCP designates these activities for the indicated number of Continuing MedicalLaboratory Education (CMLE) credits. ASCP CMLE credits are acceptable to meet the continuing education requirementsfor the ASCP Board of Registry Certification Maintenance Program. All ASCP CMLE programs are conductedat intermediate to advanced levels of learning.The primary purpose of this activity is educational and the comments, opinions, and/or recommendations expressed byauthors are their own and not those of the ASCP. The author(s) has no relevant financial relationship with commercialinterests that provide products and/or services discussed in this exercise.Materials will be replaced without charge, worldwide, if the publisher receives a request within 90 days of themailing date.For subscription information, address corrections, or notification of damaged shipments, contact the ASCP.For answer sheet instructions and the latest updates, please visit http://credit.ascp.org/. Credit for 2011Credit Check Sample for 2010 exercises Check Sample may be exercises earned through may be December earned through 31, 2013. December 31, 2012.42

HISTORYA 73-year-old woman underwent surgical repair of a ventral hernia. After anuneventful postoperative course, she was discharged on postoperative day 4. Aroundday 9, she developed progressive weakness, lightheadedness, and dark urine, and had1 episode of emesis of clear fluid. She presented to the authors’ institution on day 14.Review of all other systems was unremarkable. Her medical history was significantfor colon cancer 7 years earlier treated with surgical resection and chemotherapy,hypertension, hyperlipidemia, hypothyroidism, and gastroesophageal reflux disease.Her outpatient medications included carvedilol, nifedipine, captopril, losartanhydrochlorothiazide,ezetimibe-simvastatin, thyroxine, esomeprezole, ranitidine,aspirin, and tramadol.transfusion medicineTM 11-4© ASCP 2011Her vital signs were: blood pressure, 128/93 mmHg; pulse rate, 81/min; respirations,18/min; and temperature, 98.4°F (36.9°C). Oxygen saturation on room air was100%. Her abdominal wound was healing with no evidence of bleeding or infection.No skin rashes, petechiae, ecchymoses, or mucosal hemorrhages were present. Thefecal occult blood test result was negative. The rest of her physical examination resultswere unremarkable.Relevant admission laboratory results are shown in Laboratory Data. Thepatient had severe anemia with evidence of hemolysis, including reticulocytosis,unconjugated hyperbilirubinemia, a low haptoglobin level, an elevated level of lactatedehydrogenase, and hemoglobinemia. The peripheral smear showed abundantmicrospherocytes and no schistocytes. Her blood type was group A Rh+. Theantibody screen result was negative. The direct antiglobulin test (DAT) result waspositive for IgG and negative for C3. An acid elution was performed, and the eluatewas nonreactive with reagent red blood cells (RBCs). She was admitted for RBCtransfusions and evaluation of her hemolytic anemia.LABORATORY DATA.Test Patient Result Reference RangeWhite blood cell count, ×10 3 /µL (×10 9 /L) 5.50 (5.50) 3.28-9.29 (3.28-9.29)Red blood cell count, ×10 6 /µL (×10 12 /L) 1.96 (1.96) 3.76-4.93 (3.76-4.93)Hemoglobin, g/dL (g/L) 6.2 (62) 11.5-14.6 (115-146)Hematocrit, % 18.7 34.0-42.1Platelet count, ×10 3 /µL (×10 9 /L) 207 (207) 143-398 (143-398)Mean corpuscular volume, fL 95.4 79.0-95Absolute reticulocyte count, ×10 6 /µL (×10 9 /L) 0.1942 (194.2) 0.0273-0.1072 (27.3-107.2)Haptoglobin, mg/dL (µmol/L)

een proven. 5 There are some anecdotal reports of successful treatment of refractoryDIIHA with plasmapheresis. 10Drug-Induced Immune Thrombocytopenia (DIIT)Background and PathophysiologyIn addition to hemolytic anemia, drugs can cause thrombocytopenia throughsimilar immune-mediated processes. Heparin-induced thrombocytopenia is themost common and well described of these entities and is caused by antibodies withspecificity to complexes of platelet factor 4 and heparin. This results in moderatethrombocytopenia 5 to 14 days after treatment. Bleeding is uncommon, butthrombosis occurs in more than one half of patients. 11 DIIT due to other drugs is rareand is associated with more severe thrombocytopenia and increased risk of bleeding.The incidence of DIIT is estimated at 10 persons per million per year, thoughthis is likely an underestimate. 12 The data of George and colleagues, who reviewedcases of DIIT reported through 2008, is available online: http://www.ouhsc.edu/platelets. According to their criteria (explained in detail at the website), 51 drugs aredefinite and 17 others are probable causes of DIIT. Based on their analysis, DIITis most commonly associated with quinine and sulfonamide antibiotics. Quinidine,nonsteroidal anti-inflammatory drugs, rifampin, vancomycin, anticonvulsants, goldsalts, antineoplastics (fludarabine, oxaliplatin), and platelet inhibitors (tirofiban,eptifibatide, and abciximab) are also implicated. 12transfusion medicineTM 11-4© ASCP 2011DIIT is most commonlyassociated with quinineand sulfonamideantibiotics.Several pathophysiologic mechanisms have been proposed. An early model suggestedthat drugs bind covalently to platelet membrane proteins and act as haptens to induceformation of drug-dependent antibodies in a process analogous to DIIHA causedby high-dose penicillin. This may account for the rare cases of thrombocytopeniacaused by penicillin, piperacillin, and cephalosporins, but does not explain DIITcaused by other drugs. 12 For more than 20 years, the major hypothesis was the“immune complex” mechanism, which proposed that some drugs can react directlywith antibodies to produce immune complexes that target platelets for destruction.However, these complexes have never been demonstrated experimentally, and thishypothesis has since fallen out of favor. 12 A newer model suggests that drug-dependentantibodies are probably derived from naturally occurring autoantibodies with weakaffinity for platelet membrane glycoproteins. The presence of certain drugs improvesthe binding of the antibody to the platelet membrane glycoprotein (GP; typicallyGPIIb-IIIa or GPIb-V-IX). Examples of such drugs include quinine, sulfonamideantibiotics, nonsteroidal anti-inflammatory drugs, and anticonvulsants. 12Some drugs induce formation of true autoantibodies that can bind directly to plateletsin a drug-independent manner. One to 2% of patients treated with gold salts developplatelet autoantibodies. 12,13 Autoantibodies can also arise in response to L-dopa,procainamide, penicillamine, interferon α and β, and occasionally sulfonamideantibiotics. 12,13 This form of DIIT is clinically indistinguishable from idiopathicthrombocytopenic purpura and is diagnosed on clinical grounds.Several novel DIIT mechanisms have been described and are worth noting. Fibansare a class of platelet inhibitor drugs that includes tirofiban and eptifibatide. Theywork by binding to the recognition site on GPIIb-IIIa, preventing its interaction47

transfusion medicineTM 11-4© ASCP 2011Factors that favor DIITinclude: unexplainedacute thrombocytopeniafollowing any drugadministration, severethrombocytopenia(a platelet count of

on discovery that cefotetan and cefazolin were administered prophylactically forsurgery. To evaluate for possible DIIHA, the patient’s serum and eluate were sentto the American Red Cross Reference Laboratory (Pomona, CA) with the followingresults: The patient’s undiluted serum caused complete hemolysis of cefotetan-coatedRBCs; a 1:100 dilution of serum strongly agglutinated cefotetan-coated RBCs; theeluate also reacted with cefotetan-coated RBCs; all test results of non–drug-coatedRBCs were negative; and parallel test results for cefazolin drug-dependent antibodieswere negative. The findings confirmed the diagnosis of DIIHA due to cefotetan.Furthermore, the in vitro lysis of cefotetan-coated RBCs supported the suspicion thatintravascular hemolysis had occurred in vivo. Cefotetan DIIHA typically presentswith IgG-mediated extravascular hemolysis, which the patient clearly had, basedon the positive IgG, unconjugated hyperbilirubinemia, and the microspherocyteson the peripheral smear. Occasionally, cefotetan DIIHA involves complementmediatedintravascular hemolysis producing a DAT positive for C3. 3 The evidenceof intravascular hemolysis in the patient included hemoglobinemia, nearly absenthaptoglobin, and a very high lactate dehydrogenase level. Although C3 was notdetected in the patient’s DAT, the authors suspect that this was due to total hemolysisof the complement-coated RBCs in vivo.transfusion medicineTM 11-4© ASCP 2011Despite the patient’s severe anemia, she recovered after transfusion of several units ofRBCs and was discharged in stable condition. After the diagnosis of cefotetan DIIHAwas confirmed, she was advised to avoid cefotetan, and her drug allergy list wasupdated.REFERENCES1. Petz L, Garratty G. Immune HemolyticAnemia. 2nd ed. Philadelphia, PA:Churchill Livingston: 2004.2. Berentsen S, Beiske K, Tjønnfjord G.Primary chronic cold agglutinin disease: anupdate on pathogenesis, clinical featuresand therapy. Hematology. 2007;12(5):361–370.3. Ogose T, Wakata Y, Kaneko M, et al.A case of recurrent paroxysmal coldhemoglobinuria with the differenttemperature thresholds of Donath-Landsteiner antibodies. J Pediatr HematolOncol. 2007;29(10):716-719.4. Viraghavan R, Chakravarty A, Soreth J.Cefotetan-induced haemolytic anaemia. Areview of 85 cases. Adv Drug React ToxicolRev. 2002;21:101-107.5. Garratty G. Drug-induced immunehemolytic anemia. Hematology Am SocHematol Educ Program. 2009:73-79.6. Salama A. Drug-induced immunehemolytic anemia. Expert Opin Drug Saf.2009;8(1):73-79.7. Garratty G, Arndt PA. An update ondrug-induced immune hemolytic anemia.Immunohematology. 2007;23(3):105-119.8. Johnson ST, Fueger JT, Gottschall JL.One center’s experience: the serologyand drugs associated with drug-inducedimmune hemolytic anemia-a new paradigm.Transfusion. 2007;47(4):697-702.9. Arndt PA, Garratty G. The changingspectrum of drug-induced immunehemolytic anemia. Semin Hematol.2005;42(3):137-144.10. Garratty G. Drug-induced immunehemolytic anemia. Clin Adv Hematol Oncol.2010;8(2):98-101.11. Arepally GM, Ortel TL. Heparin-inducedthrombocytopenia. Annu Rev Med.2010;61:77-90.12. Aster RH, Curtis BR, McFarland JG, et al.Drug-induced immune thrombocytopenia:pathogenesis, diagnosis, and management.J Thomb Haemost. 2009;7(6):911-918.13. Aster RH, Bougie, DW. Drug-inducedimmune thrombocytopenia. N Engl J Med.2007;357(6):580-587.49

transfusion medicineTM 11-4© ASCP 201114. Andres E, Dali-Youcef N, Serraj K,et al. Recognition and managementof drug-induced cytopenias: theexample of idiosyncratic drug-inducedthrombocytopenia. Expert Opin Drug Saf.2009;8(2):183-190.15. Kenney B, Stack G. Drug-inducedthrombocytopenia. Arch Pathol Lab Med.2009;133(2):309-314.16. Visentin GP, Liu CY. Drug-inducedthrombocytopenia. Hematol Oncol ClinNorth Am. 2007;21(4):685-696.50

CME DOCUMENTATION QUESTIONS1. Which of the following sets of laboratory data is most consistent with immunemediatedhemolysis?A) Hemoglobin (Hg), 8.2 g/dL (11.5-14.6); total bilirubin,4.5 mg/dL (0.2-1.1 mg/dL); conjugated bilirubin, 3.9 mg/dL(0.0-0.2 mg/dL); lactate dehydrogenase (LDH), 305 U/L (91-223 U/L);haptoglobin, 35 mg/dL (30-190 mg/dL); absolute reticulocyte count,0.0513 ×10 6 /µL (0.0273-0.1072 ×10 6 /µL); direct antiglobulin test (DAT),1+ (weak) IgGB) Hg, 6.6 g/dL; total bilirubin, 3.1 mg/dL; conjugated bilirubin,0.3 mg/dL; LDH, 1703 U/L; haptoglobin,

transfusion medicineTM 11-4© ASCP 20114. A 42-year-old woman presents with thrombocytopenia (platelet count24,000/µL) 1 week after starting quinine for nocturnal muscle cramps.Drug-induced immune thrombocytopenia (DIIT) is suspected. Which of thefollowing is true?A) Detection of drug-induced antibodies (by flow cytometry) is oftendiagnostic.B) DIIT is unlikely because platelet counts are rarely less than 50,000/µL.C) DIIT is unlikely because the interval between drug administration andthrombocytopenia is too short.D) The diagnosis of DIIT due to quinine is clinical because it cannot bedistinguished from idiopathic thrombocytopenic purpura by laboratorytests.5. A 25-year-old woman presents with purpura and epistaxis 8 days after startingtrimethoprim-sulfamethoxazole for cystitis. A complete blood cell countdemonstrates a platelet count of 45,000/µL. After the diagnosis of DIIT isconfirmed, you advise the patient thatA) Upon cessation of the drug, at least 3 weeks are required for the plateletcount to return to normal.B) Platelet transfusions will reduce her mortality.C) Plasma exchange hastens recovery.D) Corticosteroids have no proven benefit.Note: To receive CME credit, please submit your answers online. To view answer sheetinstructions and updates, please visit http://www.ascp.org/checksampleinstructions. Welook forward to bringing you better results with online answer submission and certificatesthat can be printed immediately upon course completion!52