Bone Marrow Disease and Sampling in Dogs and Cats - PowerPoint Presentation

1. Bone Marrow Disease and Sampling in Dogs and CatsPart 2Wendy Blount, DVM

2. Slide PreparationPipette flecks out of the petri dish and put on glass slides immediatelyElevate one end of the slide to let extra blood run off

3. Slide PreparationPipette flecks out of the petri dish and put on glass slides immediatelyElevate one end of the slide to let extra blood run offPush spicules back up to the top of the slide, as the blood drains off

4. Slide PreparationPrepare gentle horizontal smears as well as vertical pull apart prepsLymphoma cells are fragile and require vertical prepsUse a very light touch for the horizontal preps

5. Slide PreparationDry and stain a few slides to look for bone marrow cellsYou’ll find the marrow in the clear spot on the unstained slides (displaces blood)Dark spot on the stained slides

6. Slide PreparationKeep unstained slides to submit with stained slide, in case special stains or restaining is neededWork quickly, to prevent clottingIf clots form in the petri dish, you may be able to submit clotted samples in formalin for histopathDifficult to confirm adequate sample on theseAfter confirming adequate sampling, remove the needle, and apply pressure for 30 seconds

7. Slide PreparationKeep unstained slides to submit with stained slide, in case special stains or restaining is neededWork quickly, to prevent clottingIf clots form in the petri dish, you may be able to submit clotted samples in formalin for histopathDifficult to confirm adequate sample on theseAfter confirming adequate sampling, remove the needle, and apply pressure for 30 secondsGlue incision if needed

8. AnticoagulantEDTA or citrate preferred to heparin – better cytologyNa-EDTA – add 2-3 ml saline to 10 ml purple top tube, make up new each timeK-EDTASodium Citrate 4% - JorVet J0521, Covetrus 056422Li-HeparinNa-Heparin (1000 USP) – can use in a pinch

9. Aspiration or Core Biopsy?Advantages of aspirationCellular morphology better (EDTA > heparin)Better identification of cell lineagesCharacteristics of malignancyCan calculate M:E ratiosEstimate regenerative responsesInterpret with respect to CBC and reticulocyte countNormal 0.75-2.5:1 in dogs; 1.2-2.2:1 in catsMaturation sequence counts are easierMore mature cell stages should be present in successively greater numbers (pyramid)More younger cells means leukemia, maturation arrest or immune mediated destruction of the next stage

10. Aspiration or Core Biopsy?Advantages of aspirationCellular morphology better (EDTA > heparin)Better identification of cell lineagesCharacteristics of malignancyCan calculate M:E ratiosEstimate regenerative responsesInterpret with respect to CBC and reticulocyte countNormal 0.75-2.5:1 in dogs; 1.2-2.2:1 in catsMaturation sequence counts are easierMore mature cell stages should be present in successively greater numbersMore younger cells means leukemia, maturation arrest or immune mediated destruction of the next stage

11. Aspiration or Core Biopsy?Advantages of core biopsy (take at least 2)If repeated attempts to aspirate produce no fluid (“packed marrow”)– Myelophthisic diseaseMyelofibrosis Myelonecrosis (bone marrow toxicity)If repeated attempts to aspirate produce blood only with flecks of fatAplastic anemia (hypocellular marrow)Can better evaluate marrow cellularityCan evaluate tissue architectureInvasion by normal looking lymphocytes indicates lymphomaAlways submit rolled cytology w/ bone marrow cores

12. Bone Marrow Core BiopsyUse Michel’s trephine – lateral iliac wingOr Jamshidi in any approachAs soon as the needle is well seated through the cortex, remove the stylet & capAdvance the needle 1-2 cm further, rotating in a single direction“stir” the needle to break loose the coreAttach cap to create vacuum to help retain coreRemove the needle rotating in a single directionRemove capPass the wire or stylet backward to pop the core out the top of the needle where the cap was

13. Bone Marrow Core BiopsyUse Michel’s trephine – lateral iliac wingOr Jamshidi in any approachAs soon as the needle is well seated through the cortex, remove the stylet & capAdvance the needle 1-2 cm further, rotating in a single direction“stir” the needle to break loose the coreAttach cap to create vacuum to help retain coreRemove the needle rotating in a single directionRemove capPass the wire or stylet backward to pop the core out the top of the needle where the cap was

14. Bone Marrow Core BiopsyUse Michel’s trephine – lateral iliac wingOr Jamshidi in any approachAs soon as the needle is well seated through the cortex, remove the stylet & capAdvance the needle 1-2 cm further, rotating in a single direction“stir” the needle to break loose the coreAttach cap to create vacuum to help retain coreRemove the needle rotating in a single directionRemove capPass the wire or stylet backward to pop the core out the top of the needle where the cap wasCore 0.75-1 cm long is sufficientCytologies can be made by rolling the core on slides, or scraping it

15. Bone Marrow Core BiopsyUse Michel’s trephine – lateral iliac wingOr Jamshidi in any approachAs soon as the needle is well seated through the cortex, remove the stylet & capAdvance the needle 1-2 cm further, rotating in a single direction“stir” the needle to break loose the coreAttach cap to create vacuum to help retain coreRemove the needle rotating in a single directionRemove capPass the wire or stylet backward to pop the core out the top of the needle where the cap wasCore 0.75-1 cm long is sufficientCytologies can be made by rolling the core on slides, or scraping itPlace cores in formalin for histopathology

16. Follow-UpTemporary lameness on the sampled leg is not unusualSeldom lasts for more than a few daysSeldom happens when iliac crest is sampledSome give an NSAID injection if appropriate for the patient

17. Submitting SamplesAlways confirm adequate marrow cells prior to shipping2-4 unstained slides along with stained slidesMake and dry slides quicklyBone marrow deteriorates in minutesUnstained slides should probably be fixed in methanol (Diffquick #1 Fixer)Always submit same day CBC and unstained blood smear with bone marrow samplesImpossible to interpret M:E ratio without itRequest reticulocyte count also if anemic (send unstained blood smears and EDTA blood, or NMB smears if you have it)PCV <30 in the dogPCV <25 in the cat

18. Submitting SamplesDon’t ship cytologies in the same box as formalin fixed coresformalin will damage cellular uptake of stainRequest special stains/tests if indicatedPrussian blue for ironImmunohistochemistry if neoplasia of unknown lineageFeLV IFA if latent infection suspectedRBC-Ab stains for NRIMHA; Platelet-Ab stains for ITPCan submit marrow for culture if FUORed top tube and sterile swab culturetteNever EDTA – it kills bacteria

19. Submitting SamplesCSU Lymphoma Tests – to rule out leukemiaIf leukopenia & increased blastsDog – flow cytometryCat - immunocytochemistryIf leukopenia & many small lymphocytesflow cytometryBlood flow cytometry even better if lymphocytosisIf leukopenia and no clearly neoplastic cellsPARRIf hyperglobulinemia (looking for myeloma)Immunofixation on serumCheck CSU Lymphoma Testing GuideCSU Submission Form

20. Basic Bone Marrow CytologyHypo-, normo- or hypercellularAre all three cell lines present?Erythroid, myeloid, megakaryocytesIs the normal maturation pyramid present in each cell line?<5% blasts (>20-30% = neoplasia) – round nucleiErythroids >80% RBC, rubricytes & metarubricytes – gray to pink cytoplasmMyeloids >80% segs, bands & metamyelocytes – elongate to lobed nucleusWhat is the M:E ratio? (normal ~1:1-2:1)Count 100-200 erythroid and myeloid cellsDecreased, normal, or increased ironAre abnormal cells present?

21. CellularityScan the marrow area at low powerThe normal marrow has fatty areas surrounded by plentiful cellsnormocellularmarrow

22. CellularityThy hypercellular marrow has mostly cells with very few fatty areas

23. CellularityThy hypocellular marrow has mostly fatty areas with very few cells

24. E:M RatioScan the marrow area at high power, and count 100-200 marrow cellsTally each as red (E) or white (M)Erythroid line has dark blue cytoplasm which pales as the cells mature, and eventually turns orange

25. E:M RatioScan the marrow area at high power, and count 100-200 marrow cellsTally each as red (E) or white (M)Erythroid line has dark blue cytoplasm which pales as the cells mature, and eventually turns orangeMyeloid line has granular pale cytoplasm with pink nucleus that gets darker as the cell matures

26. E:M RatioScan the marrow area at high power, and count 100-200 marrow cellsTally each as red (E) or white (M)Erythroid line has dark blue cytoplasm which pales as the cells mature, and eventually turns orangeMyeloid line has granular pale cytoplasm with pink nucleus that gets darker as the cell matures

27. E:M RatioScan the marrow area at high power, and count 100-200 marrow cellsTally each as red (E) or white (M)Erythroid line has dark blue cytoplasm which pales as the cells mature, and eventually turns orangeMyeloid line has granular pale cytoplasm with pink nucleus that gets darker as the cell maturesRRRRRRW

28. E:M RatioScan the marrow area at high power, and count 100-200 marrow cellsTally each as red (E) or white (M)Erythroid line has dark blue cytoplasm which pales as the cells mature, and eventually turns orangeMyeloid line has granular pale cytoplasm with pink nucleus that gets darker as the cell maturesWRRRRWWWRWWWRRRRRRRR

29. E:M RatioScan the marrow area at high power, and count 100-200 marrow cellsTally each as red (E) or white (M)Erythroid line has dark blue cytoplasm which pales as the cells mature, and eventually turns orangeMyeloid line has granular pale cytoplasm with pink nucleus that gets darker as the cell maturesWRWWWWRW

30. E:M RatioScan the marrow area at high power, and count 100-200 marrow cellsTally each as red (E) or white (M)Erythroid line has dark blue cytoplasm which pales as the cells mature, and eventually turns orangeMyeloid line has granular pale cytoplasm with pink nucleus that gets darker as the cell maturesWRWWWWRRWW

31. Megakaryocytes Scan at low power (10x) for huge purple multi-nucleated cells (10-50 on the slide)Increased if regenerative thrombocytopenia or iron deficiencyDecreased if non-regenerative or acute severe thrombocytopenia

32. Iron StoresHemosiderin - dark brown to blue-green iron stores at low power (10x)These iron stores are increased

33. Iron StoresIron stores – seen in dog onlyCan see them reasonably well on DiffQuickIncreased with Anemia of chronic inflammatory diseaseIncluding IMHApersistent non-regenerative anemiaMultiple transfusions Decreased with IDANeed iron supplementation

34. Abnormal Bone Marrow CellsAtypical cells – characteristics of malignancy leukemia or myelodysplasiaMalignant cells in clusters – metastasisMast cell tumorsMast cells can be present in normal marrowClusters suggests neoplasiaPlasma and Mott cells – chronic antigenic stimulationEhrlichiosis or immune mediated diseaseLarge numbers may indicate plasma cell myeloma, especially of characteristics of malignancyInfectious AgentsAbnormal Cells

35. Abnormal CellsToo many eosinophilic granulocytes

36. Abnormal Cellslymphoma

37. Abnormal CellsLeishmania spp

38. Abnormal CellsHistoplasma spp

39. Abnormal CellsCytauxzoon spp

40. Abnormal CellsMyeloma

41. Abnormal CellsMast Cell Tumor

42. Interpreting reportsAtypical Cells

43. Why Same Day CBC is ImportantM:E ratio 4:1, WBC 45,000/ul, PCV 45%Myeloid hyperplasiaM:E ratio 4:1, WBC 15,000/ul, PCV 10%Erythroid hypoplasiaM:E ratio 1:1 (normal), WBC 100,000/ul, PCV 12%, hypercellular marrowErythroid and myeloid hyperplasiaM:E ratio 1:4, WBC 1,100/ul, PCV 35%Myeloid hypoplasiaM:E ratio 1:4, WBC 11,000/ul, PCV 9%Erythroid hyperplasiaM response takes 2-3 days; E response takes 3-7 days

44. What About the Monocyte Line?Monocytes released from the bone marrow in relatively low numbers and earlyE & M have two “pools” in the marrowProliferation: ~-blasts, pro-~-cytes, ~-cytesMaturation: meta-~-cytes, bands/retics, segs/RBC~ - insert “myelo” or “rubri”Monos have only a proliferation pool in the marrow – they mature in peripheral tissuesMonocytic lines are rarely seen in bone marrow samples, unless there is severe myeloid hypoplasia

45. SummaryPowerPoints.pptx, .pdf 1 slide per page, .pdf 6 slides per pageLaboratory InformationCSU – Lymphoma GuideCSU – Clinical Immunology submission form

46. AcknowledgementsChapter 2: The Complete Blood Count, Bone Marrow Examination, and Blood BankingDouglass Weiss and Harold TvedtenSmall Animal Clinical Diagnosis by Laboratory Methods, eds Michael D Willard and Harold Tvedten, 5th Ed 2012Bone Marrow Collection in the Dog and CatAndrew MackinWestern Veterinary Conference 2008

47. AcknowledgementsChapter 77: Techniques for Bone Marrow Aspiration and BiopsyLeo J “Ty” McSherryTextbook of Veterinary Internal Medicine, eds Stephen J Ettinger and Edward C Feldman, 6th Ed 2003Interpretation of Canine Bone MarrowDouglas J Weiss and Stephanie A SmithCompendium of Continuing Education for the Practicing Veterinarian Vol 24 No. 10 October 2002

48. AcknowledgementsCollection and Assessment of Canine Bone MarrowDouglas J Weiss and Stephanie A SmithCompendium of Continuing Education for the Practicing Veterinarian Vol 24 No. 9 Sep 2002Causes of Canine and Feline PancytopeniaShawn Ann Kearns and Patty EwingCompendium of Continuing Education for the Practicing Veterinarian February 2006

49. AcknowledgementsChapter 4: Leukocyte DisordersHarold Tvedten and Rose RaskinSmall Animal Clinical Diagnosis by Laboratory Methods, eds Michael D Willard and Harold Tvedten, 5th Ed 2012enging Anemia CasesCrystal Hoh, ACVIMHeart of Texas Veterinary Specialty CenterCAVMA CE