7/30/2013 Complete blood counts in your clinic: how to get the most out of your hematology analyzer Gwendolyn J. Levine, DVM, DACVP (Clinical Pathology) College of Veterinary Medicine & Biomedical Sciences, Texas A&M University Overview • • • • • • General Comments Peripheral Blood Smears Methodology Sources of Error Different Analyzers Case Examples General Comments • Training! • Daily quality control or weekly – depending on the volume of samples being run • Maintenance protocols • Proper storage of reagents • Standardized use protocols • Garbage in, garbage out 1 7/30/2013 Quality Control • Internal QC by the instrument is not enough • Yes, the instrument is working electronically, but is it working to accurately analyze samples? • Talk to the manufacturer/vendor about appropriate QC materials • Bio-Rad USA has several hematology QC materials Instrument Limitations • • • • Yes, it looks at tens of thousands of cells Cows nRBCs Optical vs. impedance PERIPHERAL BLOOD SMEARS 2 7/30/2013 Why Make Peripheral Blood Smears? • At a glance evaluation to double check instrument • Platelet clumps • White blood cell clumps • Agglutination • Toxic Change • Leukemia • nRBCs • Organisms 3 7/30/2013 Which Patients Should Have One Made? • ALL patients ideally • Need to double check your instrument • Have an idea of what you expect to see before looking at output I Don’t Have Time To Do That! • • • • • Anemic patients Thrombocytopenic patients Sick animals Leukopenic animals (<3.5 WBC/ul) Patients with >20 or 25,000 WBCs/ul WHAT COULD YOU SEE ON A SMEAR THAT WOULD MAKE A DIFFERENCE FOR A PATIENT? 4 7/30/2013 Platelet clumps Platelet Clumps Platelet Clumps • Yes, some instruments may flag that platelet clumps are present – many won’t • Any patient who is thrombocytopenic as determined by an instrument deserves to have the result double checked by examining a smear 5 7/30/2013 Agglutination Agglutination • You may be able to see it grossly • The instrument may throw an error flag saying agglutination is present • If agglutination is present, what are you going to want to look on a blood smear for anyway? Spherocytes 6 7/30/2013 Spherocytes • Formed by macrophages in the spleen removing a bit of the RBC membrane • Results in a cell that has a decreased surface area to volume ratio but no change in volume • Even though they appear to be smaller in diameter, their volume is not different • Not sure if you are seeing spherocytes? Heinz bodies Heinz Bodies • What parameter on your instrument printout is usually affected by the presence of Heinz bodies? • Are heinz bodies important for you, the practitioner, to know about? 7 7/30/2013 Eccentrocytes Eccentrocytes • Usually seen in dogs and horses, rarely in cats • Oxidative damage to the membrane, causing it to fuse together and push hemoglobin to one side of the cell Acanthocytes and Schistocytes 8 7/30/2013 Acanthocytes and Schistocytes • • • • • • Uncommon but important! Liver disease Glomerulonephritis DIC Hemangiosarcoma Cholesterol problems Mycoplasma Cytauxzoon felis 9 7/30/2013 Babesia canis nRBCs nRBCs • Not every instrument will tell you they are present! • If there are more than 5 nRBCs/100 WBCs, you need to correct the WBC downwards • Many instruments will count these guys as lymphocytes 10 7/30/2013 Toxic Change Intracellular bacteria Intracellular Histoplasma capsulatum 11 7/30/2013 Hepatozoon americanum Mast cells Mast Cells • So far, there is only one differential for a cat with circulating mast cells in the peripheral blood. • Dogs have been reported to have circulating mast cells for a variety of causes – many of them non-specific and not bad news 12 7/30/2013 Leukemia METHODOLOGY Electronic Impedance • Particles are counted and classified based on volume alone • Different lysing agents help distinguish cells • Fast 13 7/30/2013 Light Scatter – Flow Cytometry • Cells are classified based on size and complexity • Dyes can be used to differentiate cells based on staining • Can be slow Centrifugation • Only one instrument uses this methodology • Assumptions are made about the average sizes of some cells • Measures mass SOURCES OF ERROR 14 7/30/2013 Pre-Analytical Problems • • • • • • • Blood drawn from catheter Blood into wrong anti-coagulant Delayed separation of plasma from WBCs Improper sample handling or storage Interfering substances in the sample Incorrect sample labeling Inadequate patient preparation Analytical Problems • During actual performance of a laboratory assay • Improper sample aliquoting • Improper reagent handling • Incorrect analyzer usage Detection of Errors Re-run sample – suspect values better Operator error New sample – suspect values better Preanalytical error likely If suspect values are repeated on new sample Probable analytical error or results represent patient’s status • Can try another lab • • • • • • 15 7/30/2013 DIFFERENT ANALYZERS AVAILABLE Choosing a Hematology Analyzer • • • • • • • • Cost Cost per sample - #, reagents Training (course), tech support QC materials, protocols Species Sample volume requirements, time Data output User friendliness Know the Range Over Which Your Analyzer is Accurate • Range of linearity • May need to dilute sample • May need to analyze a sample a different way – low RBC fluid • Size cutoffs – static vs. floating • Sample interference concerns – Hemolysis – Lipemia 16 7/30/2013 Do I need a 5-part differential? • Granulocytes, Lymphocytes, and Monocytes VS. • Neutrophils, Eosinophils, Basophils, Lymphocytes, and Monocytes Abaxis VetScan HM5 Hematology System • Method: Impedance • 5-part differential • Species: cat, dog, horse, cow, alpaca, and llama • Can do 3-part differential on 9 other species • 50 µm sample size • 3-4 minutes to get results Abaxis VetScan HM2 Hematology System • • • • • Method: Impedance 3-part differential Dog, cat and horse for sure 25 µm sample size 2-3 minutes to get results 17 7/30/2013 Heska HemaTrue Hematology Analyzer • Method: Impedance • Counts particles based on volume – not time to pass through an orifice • 20µl sample size • 55 seconds to get results IDEXX LaserCyte Hematology Analyzer • Method: Flow cytometry • 5-part differential • Need to have 500µl of sample in a VetCollect Tube • Validated for: dogs, cats, horse, pig, and ferret • Sample run time can be up to 15 minutes IDEXX ProCyte Dx Hematology Analyzer • Method: Flow Cytometry, Impedance • Uses 30µl of EDTA blood from a 1mL VetCollect Tube • Validated for: dogs, cats, horses, cows, ferrets, rabbits, gerbils, pigs, and mini pigs • Can do body fluids for dogs, cats, horses • Results in 2 minutes? 18 7/30/2013 IDEXX VetAutoread Hematology Analyzer • Method: Centrifugation • Give granulocyte count and lymphocyte/monocyte count • Can do % reticulocytes on dog and cat blood • Measures a plateletcrit • Total sample time: ~8-10 minutes • Sample volume: 111µl EDTA blood Scil Vet abc Method: Impedance 12 µl EDTA blood 90 seconds to results 3 part differential with Eosinophil flag for: dog, cat, horse, rabbit, rat, mouse, and ferret • Does 11 species total – WBC count, RBC count, red cell parameters, and platelet count • • • • Scil Vet abc Plus • • • • Method: Impedance 10 µl EDTA blood 60 seconds to results 4 part differential – dogs, cats, horses 19 7/30/2013 EXAMPLES AND CASES Example 1 • 3 year-old, FS, American domestic shorthair referred in for thrombocytopenia • Owner is a medical student and concerned • What do you think is going on? 20 7/30/2013 Example 1 Resolution • NaCit tube drawn and submitted – Used jugular vein and experienced technician • Platelet count: 350,000/ul (OK!) • Can also draw citrate into syringe and coat it – then expel excess out and draw blood Example 2 • Adult DSH of unknown age is referred in with a markedly elevated white blood cell count: 80,000 cells/ul • Instrument differential: – 50% neutrophils – 50% lymphocytes 21 7/30/2013 Example 2 Resolution • Patient diagnosed with acute lymphoid leukemia and was euthanized Example 3 • Dog owned by two MDs is brought in for leukopenia diagnosed by rDVM • Dr. Willard is all ready to do a bone marrow aspirate and a full internal medicine work-up! 22 7/30/2013 Example 3 Resolution • Large numbers of WBC clumps were seen on the smear prepared with EDTA blood • Blood re-submitted in LiHep and the patients WBC count was within normal limits • EDTA WBC clumping is uncommon but does occur Case 1: Signalment and History • • • • • 8 year-old, MC, domestic short hair cat Anorexia Severe lethargy Lost 11 lbs. since last summer Eats 4 cups/day of Purina cat food Physical Examination • Depressed • Mildly dehydrated – sunken eyes, prolonged skin tent • Large bladder on palpation and hepatosplenomegaly 23 7/30/2013 CBC Findings • Mild*, normocytic, normochromic, nonregenerative anemia • Elevated plasma protein • Left shift to band neutrophils and metamyelocytes without a neutrophilia or a leukocytosis, there are also moderate Dohle bodies and few toxic changes • Platelets are within the reference interval 24 7/30/2013 Chemistry Panel Findings • • • • • • • Hyperglycemia w/glucosuria Hypercholesterolemia Elevated BUN*, Creatinine is still WRI Hypophosphatemia Hyperproteinemia – A & G increased Elevated ALT Hyperbilirubinemia w/bilirubinuria Chemistry Panel Findings • • • • • • Mild hypernatremia Hypokalemia Cl is on low end of normal range LOW TCO2 Elevated Anion Gap Ketonuria present 25 7/30/2013 Urinalysis Findings • USG: 1.039 • pH is at low end of reference interval – Appropriate? • Proteinuria – next test to do? • Glucosuria and ketonuria – Has the renal threshold for glucose been exceeded? • Bilirubinuria Diagnoses? • Patient has Diabetic Ketoacidosis secondary to Diabetes Mellitus • Abdominal ultrasound diagnosed chronic pancreatitis – fPLI was elevated • Probable hepatic lipidosis although no liver aspirates were performed • Home with E-tube, and on glargine insulin “Cool” Discussion Points • Oxidative damage to RBC structures • Diabetes mellitus vs. diabetic ketoacidosis 26 7/30/2013 Oxidative Damage to RBC Structures • Damage to the hemoglobin – Heinz bodies – Cats: 8 sulfhydryl groups on Hgb • Spleen is non-sinusoidal and inefficient at removal • Damage to the membrane and cytoskeleton Eccentrocytes DKA - causes • • • • • • • • • • Insulin dependent diabetes mellitus Inadequate insulin dosing or production Infection Concurrent disease that stresses the animal Estrus Medication noncompliance Lethargy and depression Stress Surgery Idiopathic (unknown causes) Case 2: Signalment • 10 year-old, MC, Bearded Collie • Hyperglobulinemia on rDVM Chemistry Panel • Activity level improved on Rimadyl • PE: Lipomas, muscle wasting in pelvic limbs, dry hair coat 27 7/30/2013 28 7/30/2013 Additional Diagnostic Tests? • Serum Protein Electrophoresis • Bone Marrow Examination Bone Marrow Bone Marrow 29 7/30/2013 Bone Marrow Bone Marrow Bone Marrow 30 7/30/2013 Serum Protein Electrophoresis Diagnosis & Outcome • Multiple Myeloma • Treated with Melphalan with recheck CBCs Case 3: Signalment • 6 year-old, FS, Labrador retriever • History of intermittent vomiting, increasing in frequency • New mass at site of previous tumor removal 31 7/30/2013 32 7/30/2013 How many are there? • Good way to scan many WBCs quickly? Is it a ruh roh? • Are there other reasons for circulating mast cells in dogs? • What about in cats? 33 7/30/2013 34 7/30/2013 Questions? 35