Korean J Lab Med 21;3:339-44 DOI 1.3343/kjlm.21.3.4.339 Case Report Diagnostic Hematology Flow-Assisted Differential Diagnosis of Hemolytic Anemia with Spherocytosis: A Case Report Dong Il Won, M.D. Department of Laboratory Medicine, Kyungpook National University Hospital, Daegu, Korea In patients with hemolytic anemia associated with spherocytosis, differential diagnosis has to be made whether the hemolysis is immune-mediated or of non-immune origin. We report a case of hereditary spherocytosis in a 12-yr-old male child, in whom flow-assisted diagnosis was made. In this case, diagnosis was not determined because routine laboratory workups for hereditary spherocytosis yielded discrepant results: positive osmotic fragility test, positive direct antiglobulin test, and normal result in the red cell membrane protein sodium dodecyl succinimide polyacrylamide gel electrophoresis. However, all flow cytometry-based tests, such as osmotic fragility, direct antiglobulin, and eosin 5-maleimide binding test, yielded results compatible with hereditary spherocytosis. Additionally, in family study, the results of eosin 5-maleimide binding test suggested his disease being hereditary. In cases with diagnostic difficulties, flow cytometry may be used as an alternative tool, which can provide additional information in the differential diagnosis of hemolytic anemia with spherocytosis. (Korean J Lab Med 21;3:339-44) Key Words : Hemolytic anemia, Hereditary spherocytosis, Flow cytometry 서 빈혈환자에서용혈소견이있고말초혈액도말에서구형적혈구가보이면우선유전구형적혈구증 (hereditary spherocytosis, HS) 을진단으로고려하게된다. HS 적혈구는적혈구막의골격이취약해서비장끈 (splenic cord) 을통과할때마다지질이유실되고표면 / 용적비가감소하여결국구형으로변형된다. 그러나, 다른기전으로적혈구막유실이일어나는질환들즉, 면역매개용혈 (immune-mediated hemolysis, 주로비장의그물내피계통이항체가부착된적혈구막을포식 ) 이나 glucose-6- phosphate dehydrogenase (G6PD) 결핍, 불안정혈색소 ( 적혈구내혈색소가변성, 침전되어비장에서적혈구막과함께제거 ) 등에서도구형적혈구가증가하므로 HS와감별진단이필요 Received : March 12, 21 Manuscript No : KJLM1-45 Revision received : June 4, 21 Accepted : July 22, 21 Corresponding author : Dong Il Won, M.D. Department of Laboratory Medicine, Kyungpook National University Hospital, 5 Samduk-dong 2-ga, Jung-gu, Daegu 7-721, Korea Tel : +82-53-42-5291, Fax : +82-53-426-3367 E-mail : wondi@knu.ac.kr ISSN 1598-6535 론 The Korean Society for Laboratory Medicine 하다 [1]. 구형적혈구가보이는용혈빈혈의감별진단을위한검사에는삼투압취약성 (osmotic fragility, OF) 검사 (OF test), 적혈구에부착된항체를검출하는항글로불린검사와 sodium dodecyl succinimide polyacrylamide gel electrophoresis (SDS-PAGE) 를이용한적혈구막단백질분석등이있다. 그러나이들은위음성이나위양성이비교적흔하다. 즉, OF test는 HS 환자의 1-2% 에서위음성을 [2], 면역매개용혈에서위양성을보일수있다. 직접항글로불린검사 (direct antiglubulin test, DAT) 는일반적으로입원환자의 DAT 양성판정예의 1-15% 에서면역매개용혈의증거를찾을수없으므로위양성인것으로알려져있다 [3]. SDS-PAGE도매우경한 HS 나무증상보인자에서민감도가낮아서, HS의약 2-3% 에서정상소견을보이는경우가있다 [4]. 본증례는말초혈액도말에서구형적혈구가매우증가하여우선 HS가의심되었으나, 면역매개용혈과의감별이요구되었던경우이다. 용혈빈혈의원인을감별하기위하여, 유세포분석법 (flow cytometry, FC) 을이용한 eosin 5-maleimide (EMA) binding test [5-8] 등의추가검사가도움이되었으므로이를보고하고자한다. 339
34 Korean J Lab Med 21;3:339-44 증례현재 12세남자환아로, 5세때수두를앓은이후 4개월간창백함과황달성공막이지속되어개인의원을방문후검사상빈혈이심하여본원으로전원되었다. 환자의부모나형은건강상특별한사항은없었다. 신체검사상빈혈성결막과황달성공막이있었고, 간종대나비종대는없었다. 당시시행한검사실소견으로, 일반혈액검사에서 ( 참고범위 ), 백혈구수 8,32/mL, 혈색소 6.9 g/dl, 적혈구용적률 19.6%, MCV 96.7 fl, MCHC 35.5% (32.-36.), RDW 19.7% (11.5-14.5), 혈소판수 36,/mL, 망상적혈구수 32.1% 이었다. 생화학검사에서, AST 45 IU/L, ALT 11 IU/L, 젖산탈수소효소 1,35 IU/L (24-48), 총빌리루빈 3.4 mg/dl, 직접빌리루빈 1. mg/dl, haptoglobin<13 mg/dl (3-2), hemopexin 6 mg/dl (5-11) 이었다. 이검사결과들에기초하여 HS 중증도를분류하면, 환자는중증 ( 혈색소 <8. mg/dl, 망상적혈구수 >1%, 빌리루빈 >3. mg/dl) 에속하였다. 최근의말초혈액도말소견은 Fig. 1과같다. 항글로불린검사는 DiaMed-ID (DiaMed, Murten, Switzerland) 를사용한원주응집법으로시행하였는데, 7년간추적한결과를 Table 1에다른검사와함께요약하였다. 초기일시적으로 DAT 양성이었다가이후계속음성이었다. 첫입원시 (5세) 항핵항체와한랭응집소검사는음성이었다. 혈청보체가 (CH5) 39.7 U/mL (23-46), 보체3(C3) 71.6 mg/dl (9-18), 보체 4(C4) 2.1 mg/dl (1-4) 이었다. OF test는 Fig. 2와같이채혈즉시와 24시간배양후혈액모두건강인대조보다증가된 OF를보였다. 적혈구막단백질 SDS-PAGE에서정상패턴을보였고, 적혈구효소검사 (G6PD, pyruvate kinase 포함 2종, 분광광도계법 ) 에서효소결핍의증거는없었다. 첫입원시용혈빈혈의원인이면역매개성임을시사하는일부소견이있으므로, 환아에게스테로이드요법 (prednisolone 2 Fig. 1. Peripheral blood smear at 12 yr of age. Microspherocytes accounts for ~5% of red cells. A few nucleated red cells and many polychromatic macrocytes are seen (Wright-Giemsa stain, 2). Table 1. Consecutive results of antiglobulin tests and other associated tests in this case Age (yr) Polyspecific IgG Antiglobulin test Direct IgM IgA C3d Indirect Hemoglobin (g/dl) (%) Spherocytes in PB smear Bilirubin (mg/dl) 5 293/365 - - - - - - 6.9 32.1 ++ 3.4 5 31/365 - - 1.1 7.1 +.9 6 31/365 + + - + - - 6. 57.4 +++ 3.8 6 42/365 - - - + - - 1.9 16. ++ 2.5 6 44/365 - - 7.8 44.5 1.7 6 126/365 - - 8.2 25.3 4.1 6 214/365 - - 5.3 42.8 +++ 2.5 1 44/365 - - 1.4 16.2 2.5 1 5/365 - - 8.9 31. +++ 4.9 1 191/365 - - 9.7 22.5 +++ 3.9 1 295/365 - - 12.7 13.7 +++ 2.5 1 347/365 - - 8.6 29.9 +++ 2.5 11 261/365 - - 9.2 13. +++ 2.5 12 47/365 - - 9.2 22.3 +++ 2.5 12 48/365 - - 9.4 24.8 +++ 4.1 Abbreviations: PB smear, peripheral blood smear; +, slight; ++, moderate; +++, severe.
Won DI, Flow Cytometry for Hemolytic Anemia With Spherocytosis 341 1 8 Immediate 24 hr incubation %Hemolysis 6 4 2 1..8.6.4.2 1..8.6.4.2 % NaCl Fig. 2. Osmotic fragility test at 1 yr of age. The patient (thick lines) showed increased osmotic fragility as compared with a healthy control (dashed lines) in both immediate and 24 hr-incubated samples. Reference ranges are indicated in thin lines. The initial osmotic fragility test performed at 6 yr of age had also showed same patterns. Hemoglobin Bilirubin : Admission Hemoglobin, g/dl or Bilirubin, mg/dl 16. 14. 12. 1. 8. 6. 4. 2.. mg/kg/day) 이시도되었다. 스테로이드요법전과 16일후를비교하면 ( 전 후 ), 혈색소 (5.8 9.6 g/dl), 빌리루빈 (3.8.9 mg/dl), 망상적혈구수 (53.5 6.8%) 등임상양상이호전되었다. 환아는이후발열, 황달, 심한무력증등용혈빈혈이심해질때마다수차례입원하여수혈요법이나스테로이드요법을받았다. 최근 2년간의용혈빈혈관련검사결과는 Fig. 3과같다. 최근 HS 진단을위하여 FC를이용한 EMA binding test가도입되었으므로, 환자가 12세인현재, 용혈의원인이면역매개성인지비면역성인지, 혹은스테로이드요법후임상양상호전이면역억제때문인지질환의자연경과인지감별하기위하여 FC를이용한세가지추가검사들을시행하였다. FC OF test는 Won과 Suh [9] 의방법으로시행하였다. 이방법은, 적혈구를생리식염수에부유시키고유세포분석기로수집 (acquisition) 하면서중간에증류수 (deionized water, DW) 를 : RBC transfusion 1. 1.5 11. 11.5 12. Age (yr) 6. 5. 4. 3. 2. 1.., % Fig. 3. Laboratory findings associated with hemolytic anemia during last 2 yr. Steroid therapy was tried in every admission. 튜브에첨가하면적혈구가용혈되기시작는데이때실시간으로수집된적혈구수에대하여 DW 첨가전후를비교하는원리이다. 전통적인 OF test와마찬가지로이방법도건강인에비하여유의하게증가한 OF를보였다 (Fig. 4). FC direct antiglobulin test (FC DAT) 는 Won 등 [1] 의방법으로시행하였다 (Fig. 5). 적혈구의 anti-igg와 anti-igm 히스토그램에서 mean fluorescence intensity (MFI) 를구하였고, 환자와함께시행한건강인 6명의 MFI의평균 +3 표준편차 (SD) 를경계치 (cutoff) 로정하였다. 환자의 MFI는이경계치보다작으므로, IgG와 IgM 둘다음성으로판정하였다. EMA binding test는 King 등 [8] 의방법으로시행하였다 (Fig. 6). 이검사는녹색형광을내는 EMA가적혈구막의 band 3나 Rh-연관단백질에결합하는성질을이용하는데 [5], 적혈구를 EMA 염색후그형광을유세포분석기 FL1 지표
342 Korean J Lab Med 21;3:339-44 1 8 Healthy 1 8 Healthy DW %Residual red cells=89% 6 6 4 4 2 2 SSC 2 4 6 8 1 2 4 6 1 1 1 Patient Patient %Residual red cells=19% 8 DW 8 FSC 6 6 4 4 2 2 2 4 6 8 1 FSC 2 4 6 1 Time (24.8 sec) Fig. 4. Flow cytometric osmotic fragility test at 12 yr of age. The events after DW spiking are indicated in red. Post DW spiking, a main subset of the patient showed both a less increased FSC and a less decreased SSC than that of a healthy control (white arrows). On the patient s FSC/SSC plot, a subsidiary subset showing largely increased FSC appears to be reticulocytes (yellow arrow on the right). The control/patient ratio of residual red cells was 4.68 (89%/19%), which was determined as increased osmotic fragility (cutoff 3.). Abbreviations: FSC, forward scatter; SSC, side scatter; DW, deionized water. Counts 5 4 3 2 1 5 4 3 2 1 Patient MFI 4.41 Healthy MFI 4.28 1 1 1 1 2 1 3 1 4 anti-igg FITC Patient MFI 4.52 Healthy MFI 4.27 1 1 1 1 2 1 3 1 4 anti-igm PE Fig. 5. Direct antiglobulin test by flow cytometry at 12 yr of age. Patient s MFIs were below the cutoff MFIs on both the anti-igg and anti-igm histogram of red cells (4.97 and 4.99, respectively), and these values are within normal limits. Abbreviation: MFI, mean fluorescence intensity. (parameter) 로측정하여 [5-8], 적혈구의 FL1 히스토그램에서 MFI를구한다. 환자검체는세번반복측정하여평균치를구하였고, 함께시행한건강인 6명의 MFI의평균 -3 SD를경계 치로정하였다. 그결과, 환자적혈구에 EMA binding이건강인보다유의하게감소한것이증명되었다. 가족조사에서환자의부, 모, 형모두전혈구계산과말초혈액도말은정상이었으나, EMA binding이정상대조보다적었고특히형은경계치미만의유의한감소를보였다. 이들은용혈빈혈이발현된적이없으므로, 상염색체열성유전인경우모두 HS 보인자로생각되었다 (Fig. 7). 이상, HS 감별진단을위한통상검사와 FC를이용한세가지추가검사를종합하면, 용혈빈혈의원인으로서면역매개성보다비면역성용혈, 즉 HS 의가능성이더높은것으로생각되었다. 고찰본증례는구형적혈구가증가된용혈빈혈환자의 HS 감별진단을위한통상검사에서 OF test 양성, DAT 양성 ( 일시적 ), 적혈구막단백질 SDS-PAGE 정상 으로일관된소견을보이지않았으나, FC를이용한추가검사에서 FC OF test 양성, FC DAT 음성, EMA binding test 양성 으로 HS에합당한결과를얻은예이다.
Won DI, Flow Cytometry for Hemolytic Anemia With Spherocytosis 343 Healthy 4 1 Healthy MFI 523 Patient A Counts 32 24 16 8 SSC SSC 1 2 4 6 8 1 R1 R1 2 4 6 8 1 FSC Patient MFI 443 1 1 1 1 2 1 3 1 4 EMA B Fig. 6. Eosin 5-maleimide (EMA) binding test at 12 yr of age. (A) Fluorescence microscopy. EMA-bound red cells show green fluorescence. (B) Flow cytometry. Patient s MFI (443) was below the cutoff (523-3 21= 46). Abbreviations: FSC, forward scatter; SSC, side scatter; MFI, mean fluorescence intensity, EMA, eosin 5- maleimide. Hemoblobin MCV MCHC EMA MFI 16.2 g/dl 92.2 fl 33.5 pg 1.43% 356 Hemoblobin MCV MCHC EMA MFI 15.1 g/dl 87.9 fl 34. pg.91% 39 Hemoblobin MCV MCHC EMA MFI *EMA MFI of Healthy (N=6) - Mean 412 - SD 28 - Cutoff 329 11.3 g/dl 85.3 fl 33.6 pg 1.55% 332 Fig. 7. Genogram of the patient s family. The results of hematologic and EMA binding test are provided. His elder brother also showed significantly decreased MFI value compared to the cutoff value in this batch of test. As EMA binding test for the patient s family was performed in a different batch from the patient, direct comparison of MFI values between the patient s in Fig. 6 and his family s in this figure is not possible. Abbreviations: EMA, eosin 5-maleimide; MFI, mean fluorescence intensity. DAT 음성면역매개용혈은온난형의약 6% 를차지한다고하고, 통상적인 DAT가검출할수있는한계량이하로 IgG가적혈구에부착되면 DAT가음성을보일수있다. 이경우 IgG를검출하는데 FC가사용될수있다고하고 [11], 본증례에서시도한 FC 를이용한 DAT는음성이었다. 본환자는비종대가없었으나, HS에서환자의 2% 미만에서비종대가없기도하고 [12], 면역매개용혈에서도비종대가흔하므로, 본증례에서비종대유무는감별진단에도움이되지못하였다. 보체 C3 저하와일시적이지만 DAT 양성은면역매개성을시사하는소견이나, 본증례와같이감소된 C3, 정상 C4는그람음성패혈증, 바이러스혈증등에의한보체계교대경로활성화와관계가있다 [13]. DAT 위양성의원인으로서우선검사실측면에서, 검체중강한응집소가위양성을유발하고, fibrin이나침전물, 오염물에 의한적혈구응괴 (clump) 를응집반응으로오인할수있고, 적절한항응고제 (EDTA) 를사용하지않은경우보체가적혈구에부착되어응집이일어날수있다 [14]. 임상적으로고감마글로불린혈증, g-globulin 정주나특정약제를투여한경우, 세균감염등에서양성반응을볼수있다 [15]. 골수에서새로나온 HS 적혈구는덜구형이고삼투압에덜취약하다. 또한철이나, 엽산, 비타민 B12 결핍이동반된경우도 HS 적혈구의특징이감춰질수있다 [1]. 본증례에서스테로이드요법후말초혈액도말상구형적혈구비율이감소하는경우도있었는데 (Table 1), 용혈위기혹은골수무형성위기후회복기에망상적혈구가증가되었기때문에구형적혈구가적게관찰된것으로보인다. Bolton-Maggs 등 [2] 이제시한 HS의진단알고리듬에대하여살펴보면다음과같다 : 1) HS가의심되는환자가가족력이있고, 비종대등의임상양상과구형적혈구, MCHC 증가, 망상적혈구수증가등의전형적인검사실소견을보인다면, 확인을위한추가검사는필요하지않다. 2) 말초혈액도말에서구형적혈구가약간보이나, 다른검사실적, 임상적, 가족력적인증거가없는경우, HS 에대하여높은예측도를가지는선별검사가필요한데, cryohemolysis 검사와 EMA binding test가권장된다. 전통적인 OF test 결과는검사의제한점을잘알고적용되어야한다. 3) 이들선별검사의결과판정이애매하다면, 적혈구막단백질에대한 SDS-PAGE가도움이될수있다. 4) 유전자검사를추가하는것은필요하지않다. 대부분 HS 환자의유전자변이는세계적으로소수의가계에국한에서국지성으로발생하기때문이다. FC에의한 OF test [9] 는전통적방법보다신속하고정량적인측정치로판정할수있는장점이있으나, 전통적방법과마찬가지로증가된구형적혈구의원인, 즉 HS 와면역매개성간감
344 Korean J Lab Med 21;3:339-44 별은불가능할것으로생각된다. 근래도입된 EMA binding test는 HS 를위한선별검사로권장되고있으나, 유세포분석기가있어야되고 EMA stock solution 제조후유효기간이 -8 에서 4개월정도로짧은것이단점이다 [7]. 본증례에서환자와환자의형둘다 EMA binding이유의하게감소하였는데, 형은임상증상이없으므로 HS 보인자로생각되었고, 이는환자의용혈빈혈이후천적인면역매개성이아닌선천적인유전성질환임을시사한다 [16]. 본증례는, 구형적혈구가보이는용혈빈혈환자에서감별진단을위한통상검사들에서일관된소견을보이지않는경우, FC를이용한추가검사들이도움이될수있음을보여주었다. 요약구형적혈구가보이는용혈빈혈환자에서용혈이면역매개성인지비면역성인지감별해야한다. 유세포분석법으로유전구형적혈구증으로진단이가능하였던 12세남자환아의증례를보고한다. 이증례에서, 유전구형적혈구증감별진단을위한통상검사들은삼투압취약성검사양성, 직접항글로불린검사양성, 적혈구막단백질 SDS-PAGE 정상으로판정이일치하지않아서진단이어려웠다. 그러나, 유세포분석법을이용한삼투압취약성검사, 직접항글로불린검사, eosin 5-maleimide binding test 모두유전구형적혈구증에합당한결과를보였다. 또한, 가족조사에서 eosin 5-maleimide binding test 결과는이질환이유전성임을시사하였다. 이와같이, 구형적혈구가보이는용혈빈혈에서감별진단이어려운경우유세포분석법은추가정보를제공하는또다른기법으로유용할것으로생각된다. 참고문헌 1. Gallagher PG, Forget BG, Lux SE. Disorders of the erythrocyte membrane. In: Nathan DG and Orkin SH, eds. Nathan and Oski s hematology of infancy and childhood. 5th ed. Philadelphia: WB Saunders, 1998:544-664. 2. Bolton-Maggs PH, Stevens RF, Dodd NJ, Lamont G, Tittensor P, King MJ; General Haematology Task Force of the British Committee for Standards in Haematology. Guidelines for the diagnosis and management of hereditary spherocytosis. Br J Haematol 24;126:455-74. 3. Garratty G. The significance of IgG on the red cell surface. Transfus Med Rev 1987;1:47-57. 4. Lee YK, Cho HI, Park SS, Ra E, Chang YH, Hur M, et al. SDS-PAGE analysis of red cell membrane proteins in hereditary hemolytic anemia. Korean J Hematol 1999;34:559-67. ( 이영경, 조한익, 박성섭, 나은경, 장윤환, 허미나등. 유전성용혈성빈혈에서 SDS-PAGE를이용한적혈구막단백성분분석. 대한혈액학회지 1999;34:559-67.) 5. King MJ, Telfer P, MacKinnon H, Langabeer L, McMahon C, Darbyshire P, et al. Using the eosin-5-maleimide binding test in the differential diagnosis of hereditary spherocytosis and hereditary pyropoikilocytosis. Cytometry B Clin Cytom 28;74:244-5. 6. King MJ, Smythe JS, Mushens R. Eosin-5-maleimide binding to band 3 and Rh-related proteins forms the basis of a screening test for hereditary spherocytosis. Br J Haematol 24;124:16-13. 7. Kedar PS, Colah RB, Kulkarni S, Ghosh K, Mohanty D. Experience with eosin-5'-maleimide as a diagnostic tool for red cell membrane cytoskeleton disorders. Clin Lab Haematol 23;25:373-6. 8. King MJ, Behrens J, Rogers C, Flynn C, Greenwood D, Chambers K. Rapid flow cytometric test for the diagnosis of membrane cytoskeleton-associated haemolytic anaemia. Br J Haematol 2;111:924-33. 9. Won DI and Suh JS. Flow cytometric detection of erythrocyte osmotic fragility. Cytometry B Clin Cytom 29;76:135-41. 1. Won DI, Jung OJ, Lee YS, Kim SG, Suh JS. Flow cytometry antibody screening using pooled red cells. Cytometry B Clin Cytom 21;78:96-14. 11. Kerr R, Rawlinson PS, Cachia PG. Direct antiglobulin test negative, non spherocytic autoimmune haemolytic anaemia. Clin Lab Haematol 2;22:365-7. 12. Boyd AS. Hereditary spherocytosis. Am Fam Physician 1989;39:1 67-72. 13. Ruddy S. Complement. In: Rose NR, de Macario EC, et al. eds. Manual of clinical laboratory immunology. 4th ed. Washington, DC: American Society for Microbiology, 1992:114-23. 14. Downes KA and Shulman IA. Pretransfusion testing. In: Roback JD, Combs MR, et al. eds. Technical manual. 16th ed. Bethesda: American Association of Blood Banks, 28:437-63. 15. Leger RM. The positive direct antiglobulin test and immune-mediated hemolysis. In: Roback JD, Combs MR, et al. eds. Technical manual. 16th ed. Bethesda: American Association of Blood Banks, 28: 499-521. 16. Mentzer WC and Lubin BH. Red cell membrane abnormalities. In: Lilleyman JS, Hann IM, et al. eds. Pediatric hematology. 2nd ed. London: Churchill Livingstone, 1999:257-83.