Methods: A single May-Grünwald-Giemsa staining protocol has been developed and evaluated for the SP1000i.

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1 The Sysmex SP1000i for Automated Bone Marrow Slide Smear Staining Eugenie F.A. Gemen, Norbert C.J. de Wit, PhD, Marie-louise P.B. van Gerven, Jacqueline de Jongh-Leuvenink, PhD (Laboratory for Clinical Chemistry and Haematology, Jeroen Bosch Hospital, s-hertogenbosch, The Netherlands) DOI: /LMK6D4LVJ6NTRCCF Abstract Background: The SP1000i is a fully automated slide smear and staining system for blood samples. This system has several advantages over manual staining methods. The use of this automated slide stainer for bone marrow slides has not been described previously. Methods: A single May-Grünwald-Giemsa staining protocol has been developed and evaluated for the SP1000i. Results: The new protocol can be used to stain blood smears, bone marrow slides, and slides of other bodily fluids. This new staining procedure gave better staining results (ie, quality, reproducibility, and safety) than the manual method for both blood and bone marrow slides. Conclusions: The introduction of the SP1000i with a single staining protocol has much improved the staining quality of smears and efficiency in our laboratory. Our laboratory examines more than 500 bone marrow preparations and more than 7,000 blood smears annually. The Sysmex SP1000i was purchased with the goal of creating a fully automated hematology laboratory. This fully automated blood smear and staining system for microscopic peripheral blood differentiation can make blood slide smears, fixate the smears, and stain the slides in a single process. Manually obtained preparations, such as bone marrow and other bodily fluids, can also be stained. Before the introduction of the SP1000i in our laboratory, blood and bone marrow aspirate smear slides were prepared manually. The slides were stained with the Midas-E slide stainer (EMD Chemicals, Darmstadt, Germany). Both bone marrow and blood slides were stained according to the commonly accepted May-Grünwald-Giemsa procedure with a methanol fixation step. The main advantage of the new stainer is automation of the process and integration of the system in a hematology track system requiring minimal intervention. Furthermore, there are improvements in quality, reproducibility, and safety. The SP1000i is linked bi-directionally to the laboratory information system (LIS). Not only does the system use information obtained from the complete blood count to adjust its settings for preparing the blood slide smear (such as hematocrit), it also allows for reflex testing based on pre-set parameters in the LIS. For example, when a low leukocyte count (<2.0 x10 9 /L) is measured by our laboratory s hematology analyzer, 2 slide smears are automatically generated. In addition to peripheral blood smears, there are many requests for differential counts and assessment of morphology of bone marrow slides in our laboratory. Preparing bone marrow slides cannot, for obvious reasons, be automated, but the staining of these slides can. The introduction of the new stainer prompted the laboratory to investigate the possibility to automate the staining of bone marrow slides and other bodily fluids slides. The introduction of automated staining of blood smears, bone marrow slides, and other bodily fluids and the development of a universal staining protocol is described below. Materials and Methods The evaluation was undertaken with blood and bone marrow preparations from routine analysis. Blood smears were made with ethylene diamine tetra-acetic acid (EDTA) anticoagulated blood. Bone marrow smears and crush preparations were prepared by hand from fresh bone marrow or after adding anti-coagulant. Touch preparation and cytospin preparations or smears from other bodily fluids were prepared without the use of an anticoagulant. Manually made slides were left to dry for at least 30 minutes at room temperature prior to beginning the staining procedure. The staining solutions used were the Sörensen working solution, May-Grünwald solution, diluted May-Grünwald solution, and diluted Giemsa solution. For the manual staining method, a Sörensen stock solution (in-house made, Table 2) was used which consisted of 0.32 moles/l KH 2 PO 4 (Merck, Darmstadt, Germany) and 0.50 moles/l Na 2 HPO 4 2H 2 O (Merck). The Sörensen working solution was made by diluting 20.0 ml of Sörensen stock solution with 4.98 liters of demineralized water resulting in a buffer with ph 7.2 ± 0.1. May-Grünwald (Merck), and Giemsa (Merck) stock solutions were purchased. The diluted May-Grünwald solution was made by mixing ml of May-Grünwald stock solution with ml of Sörensen working solution. The diluted Giemsa solution was made by mixing 15.0 ml of Giemsa stock solution with ml of Sörensen working solution. December 2009 Volume 40 Number 12 LABMEDICINE 719

2 For the SP1000i, the VHL-Sörensen stock solution was made by dissolving moles/l KH 2 PO 4 and moles/l Na 2 HPO 4 2H 2 O in demineralized water 7 (VHL is the Dutch organization for research in laboratory hematology). A working solution of the VHL-Sörensen stock solution was made by diluting 1.0 L of VHL-Sörensen stock solution with 19.0 L of demineralized water resulting in a buffer with ph 7.1 ± 0.1. The May-Grünwald and Giemsa solutions for the SP1000i were identical to the manual method. The SP1000i dilutes the May-Grünwald and Giemsa solutions prior to staining by pre-mixing the solutions with VHL- Sörensen working solution. The SP1000i also uses the VHL-Sörensen working solution to rinse the smears. As a starting point, we used the standard protocol from the manufacturer on blood smears. The manual May-Grünwald Giemsa staining method was the reference method. The manual protocol, starting protocol (ie, manufacturers), and the final protocol for blood smears and bone marrow slides can be found in Table 1. The same laboratory technicians assessed all stained slides and required improvements were discussed for optimal staining results. Results Slides were reviewed for the color of erythrocytes, leukocytes, and other cells. For leukocytes, the color and staining quality of cytoplasm, granulation, and nucleus was assessed. The same assessment was done for both blood smears and bone marrow slides. Each of the steps in the staining protocol was varied. The changes made to the staining protocol were based on experience and literature findings. 1-4 The final staining protocol can be found in Table 1. Optimization of the staining protocol began on blood smears. A comparison with the manual method was done using the manufacturer s staining protocol of the SP1000i on routine samples with the VHL-buffer (Table 1 and Table 2). The slides were assessed macroscopically and microscopically (eg, color of cells). For 31 patients with known pathology (eg, chronic lymphatic leukemia, acute myeloid leukemia, myelodysplastic syndrome, Non-Hodgkin s lymphoma, spherocytosis, etc) a comparison was completed between the manual staining method and the automated staining procedure. The staining results for both methods were very good with identical morphological conclusions. The same protocol was used for bone marrow staining to compare the manual method with the new automated staining protocol. Samples from 14 patients (2 slides per patient for both manual and automated staining methods) were used to compare the 2 methods. Staining results and quality were equally good, but a number of small changes were made to improve the staining. Modifications were validated on 13 patients, and an average of 3 smears were analyzed per patient. The modifications made to the manufacturers staining protocol are (1) a change of buffer (Table 2); (2) the use of the VHL-buffer instead of tap water for rinsing the slides after staining; (3) reducing the fixation step with May-Grünwald stock from 5 to 3.5 minutes; and (4) reducing the staining time with diluted Giemsa-stain solution from 12 to 9 minutes. The final staining protocol (Table 1) resulted in a very reproducible staining of both blood smears and bone marrow Table 1_Settings for Manual May-Grünwald-Giemsa Staining Method and SP1000i Manual May-Grünwald- Manufacturer Settings for May- Final May-Grünwald Staining Settings for SP1000i Giemsa Staining Grünwald Staining With SP-1000i as Used for Blood Smears and Bone Marrow Methanol fixation 5 min May-Grünwald stock solution (prefixation) 1 min 1 min Dry 3 sec 3 sec May-Grünwald stock solution (fixation) 5 min 3.5 min Diluted May-Grünwald solution 5 min 3 min 3 min Rinsing with water 30 sec Diluted Giemsa solution 15 min 12 min 9 min Rinsing with Sörensen buffer Short rinse. Default settings Short rinse. Default settings Wash in water 30 sec Rinsing with water 30 sec Wash in flowing water 30 sec Dry Dry at room temperature 10 min with heater at 56ºC 10 min with heater at 56ºC Note: All reagents are identical except for the buffer. Table 2_Buffers Used for Staining Manual May-Grünwald- Manufacturer Settings for May- Final May-Grünwald Staining Settings for SP1000i Giemsa Staining Grünwald Staining With SP-1000i as Used for Blood Smears and Bone Marrow Buffer In-house made Sörensen buffer ph 7.2 Sysmex Sörensen buffer ph 6.8 (not recommended) VHL Sörensen buffer ph 7.1 VHL Sörensen buffer ph 7.1 VHL is the Dutch organization for research into laboratory hematology. Downloaded 720 from LABMEDICINE Volume Number December 2009 labmedicine.com

3 smears (Images 1A, 1B, 1C, 2A, 2B, and 2C). The overall staining quality was even better and more reproducible than the manual method. Moreover, the granulation of eosinophils and basophils (Images 1A, 1D) were more pronounced, and mast cells (Image 1D) were more distinct with the new staining method. Surprisingly, we observed red cell Cabot ring inclusions (Image 2D). Other bodily fluids, such as smears or cytospins, were stained with the new staining protocol. For 16 bodily fluids the manual method was compared with the new automated staining method. Samples included bronchoalveolar lavage, continuous ambulatory peritoneal dialysis fluid, cerebrospinal fluid, synovial fluid, and pleural fluid. Microscopic findings were identical for the 2 staining methods, and the staining quality was very good for the automated method. The final protocol for both bone marrow and blood smear slides was used on a number of new cases diagnosed in the laboratory. The following pathology was seen in bone marrow examination: Precursor B- and T-cell neoplasms 1 (ie, Precursor B-lymphoblastic leukemia ); acute myeloid leukemia 26; mature B-cell neoplasms 94 (ie, chronic lymphocytic leukemia 15, lymphoma 21, plasmacel neoplasms 57, chronic T-cell malignancy 1); chronic myeloproliferative diseases 3 (chronic myelogenous leukemia 3); myelodysplastic syndromes 45, myelodysplastic/myeloproliferative diseases 20; tumor cells 4; anemia based on a deficiency of vitamin B12 or folic acid and/or iron deficiency 26. Discussion The standard settings from the manufacturer were used as a starting point for blood smear staining, followed by fine tuning on bone marrow slides. The final automated staining result had to match the manual staining closely. The Sysmex buffer with ph 6.8 was initially used but resulted in aberrant staining. 5,6 Specifically, the erythrocytes were stained orange instead of the blue-grey colored erythrocytes seen with the manual method. We therefore changed the Sysmex buffer to the VHL-Sörensen buffer with a ph of This buffer was Image 1_May-Grünwald-Giemsa staining with SP1000i. (A) Chronic Myelogenous Leukemia, bone marrow, x1000. (B) Myelodysplastic Syndrome, bone marrow, x1000. (C) Acute myeloid leukemia, bone marrow, x1000. (D) Mast cell and eosinophil, bone marrow, x1000. December 2009 Volume 40 Number 12 LABMEDICINE 721

4 Image 2_May-Grünwald-Giemsa staining with SP1000i. (A) Chronic lymphocytic leukemia, bone marrow, x1000. (B) Lymphoma, bone marrow, x1000. (C) Plasma cell neoplasm, bone marrow, x1000. (D) Cabot ring, blood, x1000. slightly different from the buffer used in the manual staining method but gave superior results. The staining times for each step in the staining protocol were optimized. The effects of each change were assessed on all cell types in several different slides, and an optimal staining protocol was thus developed. Fixation with May-Grünwald stock solution for 5 minutes instead of 3.5 minutes resulted in stronger, deeper staining of the cells, which was perceived as unpleasant by the laboratory technicians. Staining with the Giemsa stain for 12 minutes instead of 9 minutes resulted in stronger, deeper staining of the nucleus. The May-Grünwald and Giemsa staining steps were therefore shortened resulting in improved staining of the bone marrow slides. A shorter staining time resulted in a less intense staining of cells (nucleus and cytoplasm) and cell components (granulation and chromatin). The erythrocytes were still light bluegrey and the staining corresponded very well with the manual method. A fixation step with May-Grünwald stock of 3.5 minutes and a Giemsa staining step of 9 minutes was found to combine the best of both. In contrast to the manual staining procedure, no fixation step with methanol was used in the new staining protocol. This was not a part of the default staining protocol and was found not to be required for a sufficient staining result. No loss of material was detected from the slides when a fixation step was omitted. However, the May-Grünwald stock solution used for the prefixation and fixation steps does contain methanol, which may explain why there is no separate fixation step needed with methanol. Another advantage of the new staining protocol is that there is a considerable reduction in staining time, and the processing capacity of the SP1000i is increased. There are personal preferences within each laboratory with regard to the staining results. Our laboratory has extensive experience in staining blood smears and bone marrow preparations, and we feel the described protocol in Table 1 gives the optimal result for interpreting blood smears and bone marrow preparations. Small modifications to our suggested staining protocol (staining time and buffer) can be made to meet personal preferences. Optimization of buffers and staining was successful for blood smears, bone marrow preparations, and smears from other bodily fluids. Downloaded 722 from LABMEDICINE Volume Number December 2009 labmedicine.com

5 The introduction of the SP1000i with a single staining protocol has much improved the staining quality of smears and efficiency in our laboratory. Moreover, these improvements were highly reproducible. It is likely the use of a single staining protocol for bone marrow and blood is also possible on other automated staining equipment. LM 1. Bain BJ. Blood Cells: A Practical Guide. 4th ed. Hoboken, NJ: Wiley- Blackwell; Helleman PW, de Nooij EH, Overbeeke MAM, et al. Hematologie. 4th ed. Netherlands: Bohn, Scheltema & Holkema; Horobin RW, Walter KJ. Understanding Romanowsky staining. Microscopica Acta. 1987;79: Lewis SM, Bain BJ, Bates I. Dacie and Lewis Practical Haematology. 10th ed. London, UK: Churchill Livingstone Elsevier; Barranco P. Efficient utilization of the Sysmex SP-100. Sysmex J Intl. 1997;7: Hayashi M, Gauthier S, Tatsumi N. Evaluation of an automated slide preparation and staining unit Sysmex SP-100. Sysmex J Intl. 1996;6: Aanbevolen werkwijze en terminologie bij de microscopische beoordeling van het perifere bloedbeeld Dif. Uitgegeven onder auspiciën van de Vereniging voor. Hematologisch Laboratoriumonderzoek December 2009 Volume 40 Number 12 LABMEDICINE 723