Clinical and Morphological Practices in the Diagnosis Of

Bone Marrow Transplantation (2019) 54:1022–1028 https://doi.org/10.1038/s41409-018-0374-3

ARTICLE

Clinical and morphological practices in the diagnosis of transplant- associated microangiopathy: a study on behalf of Transplant Complications Working Party of the EBMT

1 2 3 4 5 Ivan S. Moiseev ● Tatyana Tsvetkova ● Mahmoud Aljurf ● Randa M Alnounou ● Janet Bogardt ● 6 7 7 8 9 Yves Chalandon ● Mikhail Yu. Drokov ● Valentina Dvirnyk ● Maura Faraci ● Lone Smidstrup Friis ● 10 11 12 13 14 Fabio Giglio ● Hildegard T. Greinix ● Brian Thomas Kornblit ● Christiane Koelper ● Christian Koenecke ● 15 16 17 18 19 Krzysztof Lewandowski ● Dietger Niederwieser ● Jakob R. Passweg ● Christophe Peczynski ● Olaf Penack ● 20 21 22 23 24 Zinaida Peric ● Agnieszka Piekarska ● Paola Erminia Ronchi ● Alicia Rovo ● Piotr Rzepecki ● 8 25 26 13 27 Francesca Scuderi ● Daniel Sigrist ● Sanna M. Siitonen ● Friedrich Stoelzel ● Kazimierz Sulek ● 28 29 30 31 32 Dimitrios A. Tsakiris ● Urszula Wilkowojska ● Rafael F Duarte ● Tapani Ruutu ● Grzegorz W Basak

Received: 29 August 2018 / Revised: 25 September 2018 / Accepted: 2 October 2018 / Published online: 25 October 2018 © Springer Nature Limited 2018

Abstract

1234567890();,: 1234567890();,: Transplant-associated thrombotic microangiopathy (TA-TMA) is a life-threatening complication of allogeneic hematopoietic stem cell transplantation (HSCT). This study evaluated clinical and morphological practices of TA-TMA diagnosis in EBMT centers. Two questionnaires, one for transplant physician and one for morphologist, and also a set of electronic blood slides from 10 patients with TA-TMA and 10 control patients with various erythrocyte abnormalities, were implemented for evaluation. Seventeen EBMT centers participated in the study. Regarding criteria used for TA-TMA diagnosis, centers reported as follows: 41% of centers used the International Working Group (IWG) criteria, 41% used “overall TA-TMA” criteria and 18% used physician’s decision. The threshold of schistocytes to establish TA-TMA diagnosis in the participating centers was significantly associated with morphological results of test cases evaluations (p = 0.002). The mean number of schistocytes reported from blood slide analyses were 4.3 ± 4.5% for TA-TMA cases (range 0–19.6%, coefficient of variation (CV) 0.7) and 1.3 ± 1.6% for control cases (range 0–8.3%, CV 0.8). Half of the centers reported schistocyte levels below 4% for 7/10 TA-TMA cases. The intracenter variability was low, indicating differences in the institutional practices of morphological evaluation. In conclusion, the survey identified the need for the standardization of TA-TMA morphological diagnosis.

Introduction other organ involvement, hypertension and complement activation in the subset of patients [1, 2]. This complication, Transplant-associated thrombotic microangiopathy (TA- despite broadened understanding of its pathogenesis [3] and TMA) is a complication of hematopoietic stem cell trans- the significant improvement in the supportive care, may be plantation (HSCT) characterized by microangiopathic associated with a high mortality exceeding 50% [4, 5]. hemolytic anemia, renal and neurologic dysfunction, or Unfortunately, even recent patient cohorts with this complication have relatively poor outcome [6]. A number of novel therapies are currently investigated for the treatment of this syndrome, including complement inhibitors and These authors contributed equally: Tapani Ruutu, Grzegorz W. Basak coagulation modulators [7–9]. Thus, timely and accurate Electronic supplementary material The online version of this article diagnosis of TA-TMA is of vital importance. (https://doi.org/10.1038/s41409-018-0374-3) contains supplementary Due to significant variability of clinical and laboratory material, which is available to authorized users. presentation and overlap with the symptoms of other HSCT * Ivan S. Moiseev complications, the diagnosis of TA-TMA is difficult. Before [email protected] the introduction of consensus statement criteria by Blood Extended author information available on the last page of the article. and Marrow Transplant Clinical Trials Network (BMT Clinical and morphological practices in the diagnosis of transplant-associated microangiopathy: a study. . . 1023

CTN) [10] and International Working Group/European the differential diagnosis of different entities. A set of digita- Leukemia Net (IWG/ELN) [11], the reported incidence of lized blood slides of 20 patients including 20 (40-fold mag- TA-TMA varied significantly from 6 to 31% [12–15]. nification) pictures per case was provided for evaluation. The However, the variability in the reported incidence has not blood slide set comprised 10 cases with TA-TMA and 10 been reduced after the publication of criteria and still controls. TA-TMA cases were diagnosed based on Interna- reaches 5 to 29% [16–18]. Also, another definition of tional Working Group/European Leukemia Net (IWG/ELN) “overall TA-TMA” was formulated by Cho et al. [19]andis criteria between 2008 and 2013 at Pavlov First Saint Peters- used in several centers. The variability of the reported burg State Medical University or Helsinki University Hospital. incidences might be due to the different criteria used, risk The 10 control blood slides were from patients with other factors like the intensity of the conditioning or graft-versus- HSCT complications (acute GVHD, hemorrhagic cystitis, host disease (GVHD) prophylaxis with a combination of veno-occlusive disease), autoimmune hemolytic anemia calcineurin and mammalian target of rapamycinm inhibitors (AIHA), untreated myelofibrosis, myelodysplastic syndrome [20] and, on the other hand, it might be related to variability and B12 deficiency anemia (Supplement 1, Table 1S). All in the practices of diagnosis. digital images study materials are available in Supplement 2. The most subjective laboratory measure in the TA- The cases were placed in a random order and blinded to the TMA criteria is the morphological evaluation of frag- investigators. The number of schistocytes and comments for mented erythrocytes. Although currently existing recom- each case were requested. The number of erythrocytes in each mendations by the International Council for picture was pre-calculated and placed in excel file with auto- Standardization in Haematology (ICSH) provide adequate matic calculation of percentage after filling in the number of guidance for the morphological diagnosis in other throm- schistocytes observed in each digital image. For each case, 2– botic microangiopathies [21], it does not contain recom- 5digitalimagesaccordingtothedensityofthebloodslides mendations for microangiopathies occurring after HSCT. were mandatory for evaluation and included a total of 1000 Aspects like significant poikilocytosis, dysplasia or dys- erythrocytes, and the rest 15–18 were optional. The study erythropoiesis due to underlying hematologic disorder, materials were circulated in 17 EBMT transplantation centers, autoimmune or alloimmune hemolysis are not covered in including 3 in Switzerland, 3 in Germany, 3 in Poland, 2 in these recommendations. There is a lack of agreement Italy,2inRussia,1inHelsinki,1inDenmark,1inAustria regarding the value of the number or percentage of and 1 in Saudi Arabia. 6/17 centers were JACIE (Joint schistocytes in the diagnosis of TA-TMA. According to Accreditation Committee of the ISCT and the EBMT)- the Zini et al. [22], TA-TMA should be suspected when accredited. the level of fragmented erythrocytes is above 1%; however, Lesesve et al. [23] reported that the mean number of Statistical analysis schistocytes after allogeneic HSCT without TA-TMA is already about 0.8%, and is slightly higher after myeloa- Descriptive statistics were to evaluate the questionnaire blative conditioning. Thus, the margin between TA-TMA answers and the number of schistocytes from evaluations and non-TA-TMA patients is very narrow, and might be of the blood slides. The mixed model was used to assess additionally blurred by the other complications of HSCT. the association between the answers in the questionnaires The present multicenter study was conducted within the and the number of schistocytes reported. Mixed model European Society for Blood and Marrow Transplantation explained percentage of schistocytes by considering (EBMT) to evaluate the practices of TA-TMA diagnosis. question as a fixedeffect,adjustedbyanotherfixed effect, Type (TA-TMA vs Control), and by adding a random effect for Question by Center. The intra-class Methods coefficient for the schistocyte within the center was 0.29 (0.16–0.54). The level of significancewassetat0.05for This multicenter study was conducted between 2015 and all tests. 2017. The centers that agreed to participate in the study received two questionnaires: one for transplant physician and one for morphologist. The questionnaire for transplant physi- Results cian included 8 questions seeking for the parameters and guidelines used to diagnose TA-TMA in the center, and three Clinical and morphological practices in the diagnosis bibliography references were provided at the end of the of TA-TMA questionnaire summarizing the current consensus on TA- TMA. The morphologists’ questionnaire included 16 ques- The transplantation physicians in 94% of the centers used tions assessing morphology concepts and its application for diagnostic criteria for the diagnosis of TA-TMA, while in 1024 I. S. Moiseev et al.

6% this complication was diagnosed based on physician decision. In 41% of centers IWG criteria are used to establish the diagnosis of TA-TMA, in 41% “overall TA- TMA” criteria by Cho et al. [19] were used and in 18% physician’s decision was used. None of the centers parti- 1 7 cipating used BMT CTN criteria. Eighty eight percent of participating centers reported that the differential diagnosis of TA-TMA is difficult. To the question of what aspect of diagnostic criteria is most often not fulfilled in the cases with suspected TA- 2 8 TMA, most of the centers (65%) reported that for IWG it is the level of schistocytes and 24% the decrease of haptoglobin. Regarding BMT CTN criteria, 59% of centers reported that renal/neurologic dysfunction criterion fi was not ful lled in case of suspect TA-TMA, 29% the 3 9 level of schistocytes and 12% the negative Coombs test (Supplement 1, Fig. 1S). During the study, another question was what level of schistocytes is sufficient to suspect and to diagnose TA-TMA: 65% of them answered that for the diagnosis of TA-TMA a percentage of 4 10 schistocyte above 4% was required, 18% of physicians answered above 1%, 12% considered above 2%, and one center considered the level of schistocytes not important for the diagnosis. To suspect TA-TMA, 18% of physicians reported that the level of fragmented erythrocytes 5 11 was not important, 29% reported it should be above 1%, 41% reported it should be above 2% and 12% said above 4%. The specialists who evaluated peripheral blood morphology were asked about their practices on schistocyte 6 12 calculation. In 60% of the participating centers schistocytes were quantified by a physician trained in diagnostics, in 13% by a laboratory technician, in another 13% by transplant physician and in one center by automated analyzer. In 73% of the centers, blood film evaluation for schistocytes is Fig. 1 Types of schistocytes present in the circulated blood slides. 1 Microcrescents or triangular cells. 2 “Helmet” cells. 3 Keratocytes. 4 ordered by the physician when TA-TMA is suspected. Microspherocytes. 5 Acanthocytes. 6 Stomatocytes. 7 Target cells Regarding the method of quantification of schistocytes, (codocytes). 8 Degmacytes (“bite cells”). 9 Sickle cell-like fragments 67% of centers used the percentage of schistocytes quanti- (drepanocytes). 10 Dacrocytes. 11 Elliptocytes/ovalocytes. 12 Other fied per 1000 erythrocytes, and 20% used 5–10 power irregular fragments fields. One center evaluates more than 1000 erythrocytes. And 71% of the centers account for the density of the blood slide (Supplement 1, Fig. 1S). 13% calculate microspherocytes. We enquired regarding We evaluated the morphological forms of abnormal red morphological differential diagnostic of TA-TMA to other blood cells that should be considered as schistocytes. clinical entities. Here, 21% of centers reported that it is Centers answered as follows: triangular cells (93% of cen- impossible to distinguish TA-TMA morphology from ters), helmet cells (93%), keratocytes (67%), micro- AIHA, 13% from MDS, 20% from poikilocytosis due to the spherocytes (53%), degmacytes (47%), stomatocytes (13%), other non-hereditary membrane abnormalities (cancer, liver acanthocytes (7%), codocytes (7%) and dacryocytes (7%). disease, etc) and 7% from hereditary anemias. Most centers None of the centers considered drepanocytes, elliptocytes or (57%) considered automated hematology analyzers not ovalocytes as schistocytes (Fig. 1). Only 67% of the centers applicable for quantification of schistocytes, 36% stated that reported that “helmet” cells should be calculated in the manual confirmation of results is required and only one absence of the other schistocyte forms, 53% of the centers center reported that automated analyzers are accurate for calculate keratocytes in the absence of the other forms and TA-TMA diagnosis. Clinical and morphological practices in the diagnosis of transplant-associated microangiopathy: a study. . . 1025

Analysis of the blood slides according to the average level of fragmented erythrocytes their laboratory provides. We have observed that in several All centers that evaluated blood slides calculated the num- centers the evaluation of blood slides consistently produced ber of schistocytes in the mandatory slides, while 5 centers levels below 2% even for several overt cases. In the centers also evaluated additional slides. The analysis of blood slides that were classified in the lowest quartile by the number of by morphologists revealed significant variability in schis- reported schistocytes, in 7/10 TA-TMA cases, the schisto- tocyte quantification. The median number of schistocytes cyte proportion was <2%. This may lead either to missing reported for TA-TMA cases was 4.3 ± 4.5% (range, 0– diagnosis of TA-TMA or to the establishment of diagnosis- 19.6%, coefficient of variation 0.7), and for control cases based physician’s decision rather than on diagnostic criteria. 1.3 ± 1.6% (range, 0–8.3%, coefficient of variation 0.8). The study also revealed that despite the published con- The quartile margins of schistocytes quantified in TA-TMA sensus by ICSH on schistocyte definition [21], it was not cases were in the range of 0.2–3.7%, 0.7–7.0% and 1.0– widely implemented. The set of abnormal forms of ery- 11.3% across 10 cases. The quartile margins of schistocytes throcytes included in the schistocyte count was different quantified in control cases were in the range of 0–0.4%, across centers. Since it was observed that the morphological 0.1–1.4% and 0.7–5.3% across 10 cases. In the lower evaluation in TA-TMA-suspected cases is done by mor- quartile the number of schistocytes counted was below 2% phologists, laboratory technicians and transplantation phy- in 7/10 of TA-TMA cases and in 10/10 control cases. In the sicians, the obvious conclusion is that there is a need for second quartile the number of schistocytes counted was clear step-by-step instructions on how to calculate schisto- below 2% in 4/10 of TA-TMA cases and in 10/10 control cytes that could be used by all of the mentioned healthcare cases. In the third quartile the number of schistocytes professionals. As in the recent studies [24], currently counted was below 2% in 1/10 of TA-TMA cases and in 8/ existing automated systems for red blood cell fragment 10 control cases. In the fourth quartile the number of calculation were not accurate, and may serve only as the schistocytes counted was below 2% in 0/10 of TA-TMA screening before morphological evaluation. cases and in 7/10 control cases. In 95% of blood slide The results of quantification from the set of the blood evaluation results, each center remained within the same or slides give an insight into the reasons for the variability of adjusted quartile (Fig. 2). morphological practices. Due to relatively low number of The answers in the questionnaires were analyzed for the centers participating, the only statistically significant dif- association with the results of the schistocyte quantification ference was observed for the centers that included micro- in the blood slides. The analysis revealed a significant spherocytes in the count. Therefore, higher percentage of association between the level the clinicians use to establish schistocytes was reported by these centers. However, this diagnosis of TA-TMA and the number of schistocytes single result does not provide sufficient information about reported by the morphologist. In the centers where <2% the reasons behind the variability to make an adequate margin is used to suspect TA-TMA, the number of schis- consensus in the future. Thus, for each case a subjective tocyte reported was lower compared to centers that use morphological evaluation was done (Supplement 1, higher margin (3.2 ± 3.9% vs 5.8 ± 3.8%, p = 0.033). A Table 1S) by quartile, and a conclusion was made on why similar situation was observed for the required schistocyte the centers reported the schistocyte within each quartile. level to establish the diagnosis <2% vs ≥2% (1.8 ± 1.6% vs Surprisingly, these conclusions were almost identical 5.4 ± 4.3%, p = 0.002). No significant association was among all TA-TMA and control cases, making it possible to observed between the answers in the questionnaire for the draw general assumptions. In brief, for TA-TMA cases the morphologist and schistocyte level from the blood slides. quantification in the two lower quartiles was because one or Non-significant difference was observed if the center more types of abnormal erythrocytes were not calculated as included microspherocytes to the total count (6.1 ± 4.5% vs schistocytes due to absence of definitions or institutional 3.0 ± 3.2%, p = 0.08). guidelines. In the third quartile the centers generally calculated all forms mentioned in the ICSH guidelines. In the highest quartile overestimation of schistocyte level was Discussion observed due to inclusion of microcytes and poikilocytes in the count. For control cases the quantification in the two The study demonstrated that there is a significant variability lower quartiles was because one or more types of schisto- in the practices of TA-TMA diagnosis on both the clinical cytes or schistocyte-like cells were not counted. In the third and morphological parts. Most importantly, we have quartile major schistocyte types and/or one type of observed that the practices of schistocyte quantification schistocyte-like cells were quantified. In the fourth quartile affect the clinical decisions. The transplant physicians, if several types of schistocyte-like cells (spherocytes, ovalo- they strongly suspect TA-TMA, might use the set of criteria cytes) were counted as schistocytes. The highest false- 1026 I. S. Moiseev et al.

TA-TMA cases Control cases

200 12

12 EBMT/ELN criteria 4 BMT CNT criteria 150 2 2 2

12 4 14 6 12 6 1 100

122 1 12 1 8 4 7 47 8 8 4 7 4 4 12 10 2 12 14 11 1 8 11 6 24 6 8 4 12 8 11 4 2 2 3 10 8 50 2 10 2 3 4 7 8 4 1 11 4 3 13 1 7 8 4 4 4 6 81 13 11 1 14 5 6 9 1 6 8 15 11 14 15 8 5 8 14 12 2 4 14 12 10 11 8 8 3 12 11 13 10 11 8 7 10 10 8 12 1 4 2 No of schisto cytes from paricipating centers 13 11 8 4 20 6 6 2 6 9 1 1 12 6 14 5 1314 12 1 4 8 11 4 12 14 7 3 11 2 7 14 14 8 11 9 1 6 2 12 5 3 5 9 7 10 1 2 6 13 10 13 142 1 10 1 3 14 2 12 10 12 10 5 12 12 11 10 6 6 14 2 10 9 7 10 9 2 14 13 9 5 6 14 10 13 9 13 103 11 89 13 10 14 5 13 151 7 11 14 6 5 7 11 5 5 10 7 10 6 14 7 3 7 3 13 14 3 7 9 7 9 7 7 13 3 3 3 5 9 13 9 3 13 6 3 9 5 9 5 7 5 9 5 0 10 3 9 3 13 3 3 3 5 14 13 9 13 7 5 5 14 12 13 14 5 9

051015 20 Cases Fig. 2 Proportion of schistocytes in the set of circulated blood slide The horizontal long dash line indicates the 4% margin used in the digital images as calculated by participating centers. Each point IWG/ELN criteria [7]. The horizontal short dash line indicates the 2% represents the promile of schistocytes reported by individual center. margin used in the BMT CTN criteria [6] The number near the circle represents the index number of the center.

Novartis and Celgene, lecturer fees from Novartis. OP has received positive results were reported for autoimmune hemolytic honoraria and travel support from Astellas, Gilead, Jazz, MSD, Neovii anemia and B12 deficiency anemia. Biotech and Pfizer. He has received research support from Bio Rad, In conclusion, this study identified the need for a con- Gilead, Jazz, Neovii Biotech, Pierre Fabre, Sanofi and Takeda. He is sensus on the morphological evaluation of blood smears in member of the advisory board to Alexion, Jazz, Gilead, MSD and Omeros. GWB is the member of the advisory board to Omeros. cases with suspected TA-TMA. The absence of morpholo- The other authors declare that they have no conflict of interest. gical standardization affects the accuracy of clinical diagnosis, which might significantly affect the results of References ongoing and future trials evaluating new TA-TMA treat- ments. The study identified current morphological issues 1. Changsirikulchai S, Myerson D, Guthrie KA, McDonald GB, that could be addressed in a future consensus process. Alpers CE, Hingorani SR. Renal thrombotic microangiopathy after hematopoietic cell transplant: role of GVHD in pathogenesis. – Acknowledgements Special thanks to Valentina Kravcova, Tatyana Clin J Am Soc Nephrol. 2009;4:345 53. Schegoleva and Larena Darmilova for the collection of blood slides for 2. Jodele S, Davies SM, Lane A, Khoury J, Dandoy C, Goebel J, the study. et al. Diagnostic and risk criteria for HSCT-associated thrombotic microangiopathy: a study in children and young adults. Blood. 2014;124:645–53. Compliance with ethical standards 3. Gloude NJ, Khandelwal P, Luebbering N, Lounder DT, Jodele S, Alder MN, et al. Circulating dsDNA, endothelial injury, and Conflict of interest ISM had received travel grants from MSD, complement activation in thrombotic microangiopathy and Novartis, Pfizer, Celgene, Takeda, BMS, consulting fees from GVHD. Blood. 2017;130:1259–66. Clinical and morphological practices in the diagnosis of transplant-associated microangiopathy: a study. . . 1027

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Afﬁliations

1 R.M.Gorbacheva Memorial Institute of Oncology, Hematology 2 Nikiforov Russian Center of Emergency and Radiation Medicine, and Transplantation, Pavlov First Saint Petersburg State Medical Saint-Petersburg, Russian Federation University, Saint-Petersburg, Russian Federation 3 Oncology Center, King Faisal Specialist Hospital and Research 1028 I. S. Moiseev et al.

Center, Riyadh, Saudi Arabia 19 Department of Hematology, Oncology and Tumor Immunology, Charité University Medicine, Campus Virchow Clinic, 4 Department of Pathology & Laboratory Medicine, King Faisal Berlin, Germany Specialist Hospital and Research Centre, Riyadh, Saudi Arabia 20 Department of Internal Medicine, University of Zagreb School of 5 Department of Hematology, Oncology and Hemostaseology, Medicine, Zagreb, Croatia University of Leipzig, Leipzig, Germany 21 Department of Hematology and Transplantology, Medical 6 Division of Hematology, Geneva University Hospitals and Faculty University of Gdansk, Gdansk, Poland of Medicine, University of Geneva, Geneva, Switzerland 22 Unit of Hematology and Bone Marrow Transplantation, IRCCS 7 National Research Center for Hematology, Moscow, Russian San Raffaele Scientific Institute, Milan, Italy Federation 23 Department of Haematology and Central Haematology 8 Hematopoietic Stem Cell Unit- Department of Hematology- Laboratory, Inselspital, Bern University Hospital, University of Oncology, Istituto G. Gaslini, Genova, Italy Bern, Bern, Switzerland 9 Department of Hematology, Rigshospitalet, University of 24 Department of Hematology, Military Institute of Medicine, Copenhagen, Copenhagen, Denmark Warsaw, Poland 10 Hematology and Bone Marrow Transplantation Unit, San Raffaele 25 Haematology and Central Haematology Laboratory, University fi Scienti c Institute IRCCS, Milano, Italy Hospital of Bern, Bern, Switzerland 11 LKH - University Hospital Graz, Graz, Austria 26 Helsinki University Central Hospital, Helsinki, Finland 12 Department of Hematology, Rigshospitalet, Copenhagen 27 Department of Hematology, Military Institute of Medicine, University Hospital, Copenhagen, Denmark Warsaw, Poland 13 Medizinische Klinik und Poliklinik I, Universitetsklinikum Carl 28 Diagnostic Hematology, University Hospital Basel, Gustav Carus der TU Dresden, Dresden, Germany Basel, Switzerland 14 Department of Hematology, Hemostasis, Oncology and Stem Cell 29 Department of Hematology, Oncology and Internal Diseases, Transplantation, Hannover Medical School, Hannover, Germany Medical University of Warsaw, Warsaw, Poland 15 Department of Clinical Chemistry and Biochemistry, Medical 30 Hematopoietic Transplantation and Hemato-oncology Section, University of Gdansk, Gdansk, Poland Hospital Universitario Puerta de Hierro Majadahonda, Madrid, Spain 16 Department of Hematology, University of Leipzig, Leipzig, Germany 31 Clinical Research Institute, Helsinki University Hospital, Helsinki, Finland 17 Division of Hematology, University Hospital Basel, Basel, Switzerland 32 Department of Hematology, Oncology and Internal Medicine, Medical University of Warsaw, Warszawa, Poland 18 Hospital Saint Antoine, EBMT Paris Office, Paris, France