0183e33995e30da3c41eef0d19244b12 animals-15-01265.pdf 74bad8029a7548ea0102e372ba7d54f7a6346cf6 animals-15-01265.pdf 8aaefce65bfdaa81091e48a9a1be6c01f375787a0e45b6426de215e592761ce9 animals-15-01265.pdf Title: Preliminary Evaluation of an Automated Blood Cell Analyzer for Its Use with Blood Samples from Rainbow Trout Oncorhynchus mykiss Subject: Hematological studies provide essential information about the health of animals, which is crucial for veterinary medicine, scientific research, and aquaculture. Automatic hematological analyzers are an alternative to manual methods, offering faster and more reliable results. The objective of this study was to validate the Sysmex XN-1000V automatic hematology analyzer for blood samples from rainbow trout (Oncorhynchus mykiss), examine the effects of two anticoagulants (K2EDTA and lithium heparin), and establish normal blood reference values for this fish species. Additionally, comparative studies were conducted between the Sysmex XN-1000V and manual methods (hemocytometer cell count and blood smear estimation), and reference intervals were established. Ninety-nine heparinized blood samples were analyzed for validation and sample stability tests. The results showed extremely good precision, with a coefficient of variation (CV) below 3% for RBCs, HGB, and HCT and less than 5% for non-RBC cells (leukocytes plus thrombocytes). However, heterophils (%) exhibited higher variability, with a CV of 15.08%. Linearity was excellent, and the carry-over was below 1% for all parameters. The sample stability test indicated that samples could be analyzed for up to 48 h when stored at 4 C and up to 24 h at room temperature. Non-RBC cells were the first to degrade over time. The automated and manual methods demonstrated good correlation and agreement, validating the analyzer’s accuracy. The effects of two anticoagulants, K2EDTA and lithium heparin, on the blood samples were also studied. Heparin was the preferred anticoagulant for routine hematological analysis of rainbow trout blood with the Sysmex XN-1000V analyzer. In conclusion, the Sysmex XN-1000V enables complete hemogram analyses to be performed quickly and accurately, standardizing techniques, harmonizing results, and providing reliable reference intervals with O mykiss blood. Keywords: fish health; hematology; hematological methods validation; blood automated analyzer; rainbow trout Author: Montse Mesalles, Meritxell Uroz, Irene Brandts, Emmanuel Serrano, Rafaela Cuenca, Josep Pastor and Mariana Teles Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25 CreationDate: Tue Apr 29 15:59:59 2025 CEST ModDate: Tue Apr 29 16:04:41 2025 CEST Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 17 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 6742644 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- AIRHKL+VnURWPalladioL Type 1 Custom yes yes yes 10 0 MFGDYR+URWPalladioL-Roma Type 1 Custom yes yes yes 16 0 CURJKJ+URWPalladioL-Bold Type 1 Custom yes yes yes 22 0 WGILSP+URWPalladioL-Ital Type 1 Custom yes yes yes 27 0 SUOVFR+URWPalladioL-BoldItal Type 1 Custom yes yes yes 32 0 QMDDXF+CMSY10 Type 1 Builtin yes yes yes 37 0 ZXDKIP+EURM10 Type 1 Builtin yes yes yes 69 0 BNBDPN+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 83 0 BNBDPO+PalatinoLinotype CID TrueType Identity-H yes yes yes 86 0 BNBDPP+PalatinoLinotype TrueType WinAnsi yes yes no 92 0 BNBEAB+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 95 0 BNBEFD+PalatinoLinotype TrueType MacRoman yes yes no 98 0 XIRVNA+EURB10 Type 1 Builtin yes yes yes 110 0 IYLPED+CMBSY10 Type 1 Builtin yes yes yes 115 0 BNBDPN+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 130 0 BNBDPO+PalatinoLinotype CID TrueType Identity-H yes yes yes 133 0 BNBDPP+PalatinoLinotype TrueType WinAnsi yes yes no 139 0 BNBEAB+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 142 0 BNBEFC+PalatinoLinotype,BoldItalic TrueType WinAnsi yes yes no 145 0 BNBEFD+PalatinoLinotype TrueType MacRoman yes yes no 148 0 BNBDPN+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 164 0 BNBDPO+PalatinoLinotype CID TrueType Identity-H yes yes yes 167 0 BNBDPP+PalatinoLinotype TrueType WinAnsi yes yes no 173 0 BNBEAB+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 176 0 BNBEFC+PalatinoLinotype,BoldItalic TrueType WinAnsi yes yes no 179 0 BNBEFD+PalatinoLinotype TrueType MacRoman yes yes no 182 0 BNBDPN+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 205 0 BNBDPO+PalatinoLinotype CID TrueType Identity-H yes yes yes 208 0 BNBDPP+PalatinoLinotype TrueType WinAnsi yes yes no 214 0 BNBEAB+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 217 0 BNBEFC+PalatinoLinotype,BoldItalic TrueType WinAnsi yes yes no 220 0 BNBEFD+PalatinoLinotype TrueType MacRoman yes yes no 223 0 BNBDPN+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 253 0 BNBDPO+PalatinoLinotype CID TrueType Identity-H yes yes yes 256 0 BNBDPP+PalatinoLinotype TrueType WinAnsi yes yes no 262 0 BNBEAA+PalatinoLinotype,Italic CID TrueType Identity-H yes yes yes 265 0 BNBEAB+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 271 0 BNBEFC+PalatinoLinotype,BoldItalic TrueType WinAnsi yes yes no 274 0 BNBEFD+PalatinoLinotype TrueType MacRoman yes yes no 277 0 BNBDPN+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 327 0 BNBDPO+PalatinoLinotype CID TrueType Identity-H yes yes yes 330 0 BNBDPP+PalatinoLinotype TrueType WinAnsi yes yes no 336 0 BNBEAB+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 339 0 BNBEFC+PalatinoLinotype,BoldItalic TrueType WinAnsi yes yes no 342 0 BNBEFD+PalatinoLinotype TrueType MacRoman yes yes no 345 0 BNBDPN+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 385 0 BNBDPO+PalatinoLinotype CID TrueType Identity-H yes yes yes 388 0 BNBDPP+PalatinoLinotype TrueType WinAnsi yes yes no 394 0 BNBEAB+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 397 0 BNBEFC+PalatinoLinotype,BoldItalic TrueType WinAnsi yes yes no 400 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2025-05-27 02:09:40 CEST RepresentationInformation: animals-15-01265.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2025-05-26 13:48:37 CEST Size: 6742644 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 595 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Materials and Methods Destination: section.2 Children: Item: Title: Animals Destination: subsection.2.1 Item: Title: Instruments and Reference Methods Destination: subsection.2.2 Children: Item: Title: Automated Hematology Analyzer Destination: subsubsection.2.2.1 Item: Title: Manual Count of Rainbow Trout Blood Samples Destination: subsubsection.2.2.2 Item: Title: Sysmex XN-1000V Validation for Rainbow Trout Blood Samples Destination: subsection.2.3 Children: Item: Title: Precision Destination: subsubsection.2.3.1 Item: Title: Linearity Destination: subsubsection.2.3.2 Item: Title: Carry-Over Destination: subsubsection.2.3.3 Item: Title: Stability Test Destination: subsubsection.2.3.4 Item: Title: Agreement Between Manual and Automated Methods Destination: subsection.2.4 Item: Title: Reference Intervals Destination: subsection.2.5 Item: Title: Statistical Analysis Destination: subsection.2.6 Item: Title: Results Destination: section.3 Children: Item: Title: Precision Destination: subsection.3.1 Item: Title: Linearity Destination: subsection.3.2 Item: Title: Carry-Over Destination: subsection.3.3 Item: Title: Stability Destination: subsection.3.4 Item: Title: Agreement Between Automated Blood Cell Analyzer and Manual Methods Destination: subsection.3.5 Item: Title: Effect of the Anticoagulant Destination: subsection.3.6 Item: Title: Reference Intervals (RIs) Destination: subsection.3.7 Item: Title: Discussion Destination: section.4 Item: Title: Conclusions Destination: section.5 Item: Title: References Destination: section.6 Info: Title: Preliminary Evaluation of an Automated Blood Cell Analyzer for Its Use with Blood Samples from Rainbow Trout Oncorhynchus mykiss Author: Montse Mesalles, Meritxell Uroz, Irene Brandts, Emmanuel Serrano, Rafaela Cuenca, Josep Pastor and Mariana Teles Subject: Hematological studies provide essential information about the health of animals, which is crucial for veterinary medicine, scientific research, and aquaculture. Automatic hematological analyzers are an alternative to manual methods, offering faster and more reliable results. The objective of this study was to validate the Sysmex XN-1000V automatic hematology analyzer for blood samples from rainbow trout (Oncorhynchus mykiss), examine the effects of two anticoagulants (K2EDTA and lithium heparin), and establish normal blood reference values for this fish species. Additionally, comparative studies were conducted between the Sysmex XN-1000V and manual methods (hemocytometer cell count and blood smear estimation), and reference intervals were established. Ninety-nine heparinized blood samples were analyzed for validation and sample stability tests. The results showed extremely good precision, with a coefficient of variation (CV) below 3% for RBCs, HGB, and HCT and less than 5% for non-RBC cells (leukocytes plus thrombocytes). However, heterophils (%) exhibited higher variability, with a CV of 15.08%. Linearity was excellent, and the carry-over was below 1% for all parameters. The sample stability test indicated that samples could be analyzed for up to 48 h when stored at 4 C and up to 24 h at room temperature. Non-RBC cells were the first to degrade over time. The automated and manual methods demonstrated good correlation and agreement, validating the analyzer’s accuracy. The effects of two anticoagulants, K2EDTA and lithium heparin, on the blood samples were also studied. Heparin was the preferred anticoagulant for routine hematological analysis of rainbow trout blood with the Sysmex XN-1000V analyzer. In conclusion, the Sysmex XN-1000V enables complete hemogram analyses to be performed quickly and accurately, standardizing techniques, harmonizing results, and providing reliable reference intervals with O mykiss blood. Keywords: fish health; hematology; hematological methods validation; blood automated analyzer; rainbow trout Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25 CreationDate: Tue Apr 29 15:59:59 CEST 2025 ModDate: Tue Apr 29 16:04:41 CEST 2025 ID: 0x6216ed844e4c5b0080ca2a602b7b7fde, 0x6216ed844e4c5b0080ca2a602b7b7fde Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: MFGDYR+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 252 FontDescriptor: FontName: MFGDYR+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: SUOVFR+URWPalladioL-BoldItal FontSubset: true FirstChar: 79 LastChar: 121 FontDescriptor: FontName: SUOVFR+URWPalladioL-BoldItal Flags: Symbolic FontBBox: -170, -300, 1073, 935 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: IYLPED+CMBSY10 FontSubset: true FirstChar: 2 LastChar: 2 FontDescriptor: FontName: IYLPED+CMBSY10 Flags: Symbolic FontBBox: -26, -940, 1332, 825 FontFile: true ToUnicode: true Font: BaseFont: QMDDXF+CMSY10 FontSubset: true FirstChar: 0 LastChar: 14 FontDescriptor: FontName: QMDDXF+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true ToUnicode: true Font: BaseFont: ZXDKIP+EURM10 FontSubset: true FirstChar: 11 LastChar: 22 FontDescriptor: FontName: ZXDKIP+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true ToUnicode: true Font: BaseFont: CURJKJ+URWPalladioL-Bold FontSubset: true FirstChar: 3 LastChar: 150 FontDescriptor: FontName: CURJKJ+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: AIRHKL+VnURWPalladioL FontSubset: true FirstChar: 160 LastChar: 225 FontDescriptor: FontName: AIRHKL+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: WGILSP+URWPalladioL-Ital FontSubset: true FirstChar: 2 LastChar: 252 FontDescriptor: FontName: WGILSP+URWPalladioL-Ital Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: XIRVNA+EURB10 FontSubset: true FirstChar: 22 LastChar: 22 FontDescriptor: FontName: XIRVNA+EURB10 Flags: Symbolic FontBBox: -30, -247, 1178, 723 FontFile: true ToUnicode: true Pages: Page: Sequence: 1 Annotations: Annotation: Subtype: Link Rect: 53, 177, 152, 186 Annotation: Subtype: Link Rect: 34, 167, 74, 174 Annotation: Subtype: Link Rect: 36, 75, 152, 84 Annotation: Subtype: Link Rect: 34, 63, 89, 72 Annotation: Subtype: Link Rect: 34, 773, 133, 809 Annotation: Subtype: Link Rect: 515, 773, 560, 809 Annotation: Subtype: Link Rect: 118, 642, 129, 655 Annotation: Subtype: Link Rect: 279, 642, 290, 655 Annotation: Subtype: Link Rect: 387, 642, 398, 655 Annotation: Subtype: Link Rect: 479, 642, 490, 655 Annotation: Subtype: Link Rect: 103, 628, 114, 641 Annotation: Subtype: Link Rect: 212, 628, 223, 641 Annotation: Subtype: Link Rect: 424, 27, 560, 37 Annotation: Subtype: Link Rect: 34, 344, 87, 363 Page: Sequence: 2 Annotations: Annotation: Subtype: Link Rect: 274, 565, 281, 574 ActionDest: 414 Annotation: Subtype: Link Rect: 284, 565, 291, 574 ActionDest: 414 Annotation: Subtype: Link Rect: 509, 538, 516, 547 ActionDest: 414 Annotation: Subtype: Link Rect: 280, 454, 287, 463 ActionDest: 414 Annotation: Subtype: Link Rect: 424, 384, 431, 393 ActionDest: 414 Annotation: Subtype: Link Rect: 541, 356, 553, 365 ActionDest: 414 Annotation: Subtype: Link Rect: 529, 273, 541, 282 ActionDest: 414 Annotation: Subtype: Link Rect: 544, 273, 556, 282 ActionDest: 414 Annotation: Subtype: Link Rect: 441, 203, 453, 212 ActionDest: 414 Annotation: Subtype: Link Rect: 453, 203, 465, 212 ActionDest: 414 Annotation: Subtype: Link Rect: 466, 203, 478, 212 ActionDest: 414 Annotation: Subtype: Link Rect: 481, 203, 493, 212 ActionDest: 417 Annotation: Subtype: Link Rect: 343, 161, 355, 170 ActionDest: 417 Annotation: Subtype: Link Rect: 356, 161, 367, 170 ActionDest: 417 Annotation: Subtype: Link Rect: 226, 120, 238, 129 ActionDest: 417 Annotation: Subtype: Link Rect: 241, 120, 253, 129 ActionDest: 417 Page: Sequence: 3 Annotations: Annotation: Subtype: Link Rect: 217, 661, 229, 670 ActionDest: 417 Annotation: Subtype: Link Rect: 229, 661, 241, 670 ActionDest: 417 Annotation: Subtype: Link Rect: 305, 393, 317, 402 ActionDest: 417 Page: Sequence: 4 Annotations: Annotation: Subtype: Link Rect: 216, 561, 223, 573 ActionDest: 66 Page: Sequence: 5 Page: Sequence: 6 Annotations: Annotation: Subtype: Link Rect: 266, 465, 278, 475 ActionDest: 417 Annotation: Subtype: Link Rect: 298, 365, 506, 377 Annotation: Subtype: Link Rect: 485, 278, 492, 290 ActionDest: 120 Annotation: Subtype: Link Rect: 531, 251, 538, 263 ActionDest: 120 Page: Sequence: 7 Annotations: Annotation: Subtype: Link Rect: 199, 463, 206, 475 ActionDest: 120 Annotation: Subtype: Link Rect: 199, 183, 206, 195 ActionDest: 152 Annotation: Subtype: Link Rect: 225, 183, 232, 195 ActionDest: 152 Annotation: Subtype: Link Rect: 334, 86, 341, 98 ActionDest: 152 Page: Sequence: 8 Page: Sequence: 9 Annotations: Annotation: Subtype: Link Rect: 360, 697, 367, 709 ActionDest: 237 Annotation: Subtype: Link Rect: 420, 697, 427, 709 ActionDest: 237 Page: Sequence: 10 Annotations: Annotation: Subtype: Link Rect: 216, 661, 223, 673 ActionDest: 305 Page: Sequence: 11 Annotations: Annotation: Subtype: Link Rect: 502, 714, 509, 726 ActionDest: 375 Annotation: Subtype: Link Rect: 212, 453, 219, 465 ActionDest: 405 Page: Sequence: 12 Annotations: Annotation: Subtype: Link Rect: 453, 302, 465, 311 ActionDest: 417 Annotation: Subtype: Link Rect: 466, 302, 477, 311 ActionDest: 417 Annotation: Subtype: Link Rect: 226, 232, 238, 241 ActionDest: 417 Annotation: Subtype: Link Rect: 296, 134, 308, 144 ActionDest: 417 Annotation: Subtype: Link Rect: 232, 120, 244, 130 ActionDest: 417 Annotation: Subtype: Link Rect: 363, 65, 374, 74 ActionDest: 417 Page: Sequence: 13 Annotations: Annotation: Subtype: Link Rect: 459, 703, 471, 712 ActionDest: 417 Annotation: Subtype: Link Rect: 323, 661, 335, 670 ActionDest: 417 Annotation: Subtype: Link Rect: 335, 661, 347, 670 ActionDest: 417 Annotation: Subtype: Link Rect: 530, 633, 542, 642 ActionDest: 417 Annotation: Subtype: Link Rect: 457, 577, 469, 586 ActionDest: 417 Annotation: Subtype: Link Rect: 472, 577, 484, 586 ActionDest: 417 Annotation: Subtype: Link Rect: 190, 564, 202, 572 ActionDest: 420 Annotation: Subtype: Link Rect: 420, 536, 432, 545 ActionDest: 417 Annotation: Subtype: Link Rect: 334, 368, 346, 377 ActionDest: 420 Annotation: Subtype: Link Rect: 206, 354, 218, 364 ActionDest: 420 Annotation: Subtype: Link Rect: 530, 215, 542, 224 ActionDest: 417 Annotation: Subtype: Link Rect: 323, 188, 330, 196 ActionDest: 414 Annotation: Subtype: Link Rect: 465, 187, 477, 196 ActionDest: 420 Annotation: Subtype: Link Rect: 478, 187, 490, 196 ActionDest: 420 Annotation: Subtype: Link Rect: 549, 90, 556, 99 ActionDest: 414 Page: Sequence: 14 Annotations: Annotation: Subtype: Link Rect: 232, 675, 244, 684 ActionDest: 420 Annotation: Subtype: Link Rect: 247, 675, 259, 684 ActionDest: 420 Annotation: Subtype: Link Rect: 393, 647, 405, 656 ActionDest: 414 Annotation: Subtype: Link Rect: 406, 647, 418, 656 ActionDest: 420 Annotation: Subtype: Link Rect: 449, 619, 461, 628 ActionDest: 420 Annotation: Subtype: Link Rect: 207, 591, 219, 600 ActionDest: 417 Annotation: Subtype: Link Rect: 333, 577, 345, 586 ActionDest: 420 Annotation: Subtype: Link Rect: 235, 452, 242, 461 ActionDest: 414 Annotation: Subtype: Link Rect: 243, 452, 255, 461 ActionDest: 414 Annotation: Subtype: Link Rect: 255, 452, 267, 461 ActionDest: 414 Annotation: Subtype: Link Rect: 268, 452, 280, 461 ActionDest: 420 Annotation: Subtype: Link Rect: 280, 452, 292, 461 ActionDest: 420 Annotation: Subtype: Link Rect: 293, 452, 305, 461 ActionDest: 420 Annotation: Subtype: Link Rect: 329, 424, 341, 433 ActionDest: 420 Annotation: Subtype: Link Rect: 335, 396, 347, 405 ActionDest: 417 Annotation: Subtype: Link Rect: 434, 341, 446, 350 ActionDest: 417 Annotation: Subtype: Link Rect: 447, 341, 459, 350 ActionDest: 417 Annotation: Subtype: Link Rect: 459, 341, 471, 350 ActionDest: 417 Annotation: Subtype: Link Rect: 472, 341, 484, 350 ActionDest: 420 Annotation: Subtype: Link Rect: 484, 341, 496, 350 ActionDest: 420 Annotation: Subtype: Link Rect: 470, 313, 482, 322 ActionDest: 420 Page: Sequence: 15 Annotations: Annotation: Subtype: Link Rect: 505, 579, 542, 590 Annotation: Subtype: Link Rect: 59, 542, 96, 552 Annotation: Subtype: Link Rect: 102, 542, 138, 552 Annotation: Subtype: Link Rect: 485, 528, 522, 539 Annotation: Subtype: Link Rect: 385, 478, 422, 488 Annotation: Subtype: Link Rect: 109, 453, 146, 463 Annotation: Subtype: Link Rect: 461, 426, 498, 437 Annotation: Subtype: Link Rect: 367, 375, 405, 386 Annotation: Subtype: Link Rect: 469, 350, 506, 361 Annotation: Subtype: Link Rect: 336, 324, 373, 335 Annotation: Subtype: Link Rect: 517, 299, 554, 310 Annotation: Subtype: Link Rect: 59, 261, 96, 271 Annotation: Subtype: Link Rect: 409, 209, 447, 220 Annotation: Subtype: Link Rect: 59, 172, 96, 182 Annotation: Subtype: Link Rect: 356, 145, 394, 156 Page: Sequence: 16 Annotations: Annotation: Subtype: Link Rect: 109, 731, 146, 742 Annotation: Subtype: Link Rect: 152, 731, 188, 742 Annotation: Subtype: Link Rect: 59, 693, 96, 703 Annotation: Subtype: Link Rect: 506, 654, 543, 665 Annotation: Subtype: Link Rect: 59, 629, 96, 639 Annotation: Subtype: Link Rect: 417, 603, 455, 614 Annotation: Subtype: Link Rect: 331, 526, 369, 537 Annotation: Subtype: Link Rect: 140, 489, 177, 499 Annotation: Subtype: Link Rect: 95, 450, 133, 461 Annotation: Subtype: Link Rect: 159, 412, 197, 422 Annotation: Subtype: Link Rect: 318, 386, 355, 397 Annotation: Subtype: Link Rect: 221, 360, 258, 371 Annotation: Subtype: Link Rect: 432, 335, 469, 346 Annotation: Subtype: Link Rect: 347, 284, 385, 295 Annotation: Subtype: Link Rect: 95, 259, 133, 269 Annotation: Subtype: Link Rect: 423, 233, 460, 244 Annotation: Subtype: Link Rect: 215, 195, 252, 205 Annotation: Subtype: Link Rect: 520, 182, 557, 193 Annotation: Subtype: Link Rect: 292, 156, 329, 167 Annotation: Subtype: Link Rect: 442, 130, 480, 141 Annotation: Subtype: Link Rect: 486, 130, 522, 141 Annotation: Subtype: Link Rect: 387, 79, 425, 90 Page: Sequence: 17 Annotations: Annotation: Subtype: Link Rect: 261, 743, 299, 754 Annotation: Subtype: Link Rect: 446, 718, 483, 729 Annotation: Subtype: Link Rect: 279, 679, 317, 690 Annotation: Subtype: Link Rect: 351, 629, 389, 639 Annotation: Subtype: Link Rect: 485, 603, 522, 614 Annotation: Subtype: Link Rect: 512, 577, 550, 588 Annotation: Subtype: Link Rect: 271, 552, 309, 563 Annotation: Subtype: Link Rect: 518, 526, 555, 537 Annotation: Subtype: Link Rect: 59, 502, 96, 512 Annotation: Subtype: Link Rect: 102, 502, 138, 512 Annotation: Subtype: Link Rect: 142, 476, 180, 486 Annotation: Subtype: Link Rect: 258, 437, 295, 448 Annotation: Subtype: Link Rect: 301, 437, 337, 448 Annotation: Subtype: Link Rect: 216, 411, 253, 422 Annotation: Subtype: Link Rect: 95, 387, 133, 397 Annotation: Subtype: Link Rect: 266, 360, 303, 371 Checksum: a3030a2b Type: CRC32 Checksum: 0183e33995e30da3c41eef0d19244b12 Type: MD5 Checksum: 74bad8029a7548ea0102e372ba7d54f7a6346cf6 Type: SHA-1 Checksum: 8aaefce65bfdaa81091e48a9a1be6c01f375787a0e45b6426de215e592761ce9 Type: SHA-256