Review hematology diagnosis in veterinary

Review hematology diagnosis in veterinary

1. Hematology Diagnostic Review

2. Hematology-BASICS: Blood: Blood is fluid connective tissue present in circulatory system. Blood is the fluid of life, growth and health. It has following composition.

3. 1. Erythrocyte, leucocyte evaluation: Indications: Tachycardia Fatigue Shortness of breath Un explained jaundice Pallor conjunctiva or mucous membranes Glossitis, gingivitis, stomatitis, hematemesis, hematochezia, and many more Protocol of Manual RBC, WBC Count: Neubauer’s chamber is used for both types of counts. Diluting fluid however is mostly isotonic saline solution or Hayem’s solution for RBC counting and Turk’s fluid for WBC counting. RBC Counting Area The large center square is used for RBC counts. As already stated, this area is subdivided into 25 medium squares, which in turn are each divided into 16 squares. Of the 25 medium sqaures , only the four corner squares and the center square within the large center square are used to perform RBC counts.

4. WBC Counting Area The four large squares placed at the corners are used for white blood cell count. Since their concentration is lower than red blood cells a larger area is required to perform the cell count. Platelet Counting Area The large center square is used to count platelets. Platelets in all 25 squares within the large center square are counted. Calculations. Inferences: RBC Disorders: Polycythemia Anemia Erythrocytoses Abnormal RBCs

5. Abnormal rbcs morphology: Classed on the basis of: Color Polychromatic (Hemolytic anemia) Hypo chromatic (Fe-deficit anemia) Size Macrocytes (Internal hemorrhage) Microcytes (Fe-deficit anemia) Shape Acanthocytes , eccentrocytes,elliptocytes , etc.

6. Conti. Structures on/in RBCs Basophilic stippling, nucleated RBCs Infectious agents Arrangement of cells on blood films Rouleaux formation Agglutination Infectious agents in RBCs Babesia,Theileria , Anaplasma , Distemper inclusions, Haemobartonella species, Bartonella species, etc.

7. hCT / pcv and TS (Total Solids): When heparinized blood is centrifuged, the red blood cells become packed at the bottom of the tube, while the plasma is left at the top as a clear liquid. The ratio of the volume of packed red cells to the total blood volume is called the hematocrit.

8. Clinical interpretation: Increased PCV indicates dehydration Decreased PCV indicates RBC destruction or lack of adequate production or blood loss. Packed Cell Volume % Total Solids g/dl Rule Out ↑ ↑ dehydration ↑ normal dehydration, protein loss ↑ ↓ acute blood loss normal ↑ anemia, dehydration normal normal normal normal ↓ protein loss ↓ ↑ anemia, dehydration ↓ normal RBC destructive disease ↓ ↓ whole blood loss

9. Hemoglobin determination: Clinical Interpretation: Hb measure is important measure for anemia and polycythemia diagnosis.

10. BLOOD CELL INDICES (MCV, MCH,MCHC) Diagnosis of the type of anemia may be assisted by relating the measurements of red blood cell count, hematocrit and hemoglobin to derive the mean corpuscular volume (MCV= Average volume of RBCs), the Hemoglobin amount per red blood cell ( MCH )  and the amount of hemoglobin relative to the size of the cell, per red blood cell- the mean corpuscular hemoglobin concentration (MCHC). Erythrocytes that have a normal size or volume (normal MCV) are called normocytic , When the MCV is high, they are called macrocytic. When the MCV is low, they are termed microcytic.

11. Erythrocyte sedimentation rate (ESR) The ESR is a simple non-specific screening test that indirectly measures the presence of inflammation in the body. It reflects the tendency of red blood cells to settle more rapidly in the face of some disease states, usually because of increases in plasma fibrinogen, immunoglobulins, and other acute-phase reaction proteins.

12. Some interferences which increase ESR: increased level of fibrinogen, gamma globulins. technical factors: tilted ESR tube, high room temperature. Some interferences which decrease ESR: abnormally shaped RBC (sickle cells, spherocytosis). technical factors: short ESR tubes, low room temperature, delay in test performance (>2 hours), clotted blood sample, excess anticoagulant, bubbles in tube.

13. Modern technology for HAEMATOLOGY: XP-300 Hematology Analyzer Pre-dilute mode: 8 Parameters, which includes WBC, RBC, HGB, HCT, MCV, MCH, MCHC, PLT. Whole blood mode: 17 parameters with 3-part Differential count. Efficiently processes 60 samples/hour. Can store data of 40,000 complete blood analysis data bases.

14. WBC COUNT (total and differential):

15. Clinical interpretation: Leucocytosis has following types: Neutrophilia (high neutrophil count) often signals localized infections e.g. inflammation, bacterial infection, acute stress, steroid effects, etc. Eosinophilia (high eosinophil count) may indicate allergic and hypersensitivity conditions or invasion by parasites. Lymphocytosis (high lymphocyte count) may be seen in some viral infections. Monocytosis seen in some bacterial infections (pyogranulomatous lesions), hemolysis, hemorrhage. Basophilia usually accompanied by eosinophilia. Seen in allergic reactions, drug incompatibility reactions, neoplasms and post operative complication of spleenactamy .

16. Clinical interpretation: Leucopenia includes: Neutropenia (low neutrophil count) occurs in typhoid fever, or infectious hepatitis, Eosinopenia (low eosinophil count) may be produced by an elevated secretion of corticosteroids (in states of stress). Basopenia and Monocytopenia have rare clinical manifestations as well as diagnosis. Leukemia is a disease characterized by the progressive overproduction of WBCs which usually appear in the circulating blood in an immature form.

17. PLATELET DISOREDERS: Platelets or thrombocytes are small colorless, non-nucleated and moderately refractive bodies. Important Properties include: Adhesiveness Aggregation Agglutination DISORDERS: Thrombocytopenia acute infections, acute leukemia, aplastic anemia, pox, splenomegaly, typhoid, Tuberculosis

18. Thrombocytosis- allergic conditions, hemorrhage, bone fracture, surgical operations, trauma.

19. THICK PERIPHERAL BLOOD FILM: This is prepared for detecting blood parasites such as malaria and microfilaria. Procedure: Place a large drop of blood in the centre of a clean glass slide. Spread it in a circular area of 1.5 cm with the help of a stick or end of another glass slide. Dry Staining

20. FIELD APPLICATIONS OF BLOOD SMEARS-HAEMOPARASITES: Trypanosomiasis (T. equiperdum ) Babesiosis (B. CANIS) OF DOGS

21. Anaplasmosis (A. MARGINALE) THEILERIOSIS (T.PARVA)

22. HAEMOPROTEUS IN BIRDS LEUCOCYTOZOON IN AVES

23. Other examples of use of blood smear: Hypochromic microcytic anemia Leukemia

24. Bacteremia: Staphylococcal bacteremia Yersinia pestis (plague)

25. References: Guyton and Hall, textbook of veterinary physiology, 11th edition. K. sembulingam , Essentials of medical physiology, 3rd edition. Letimerr’s textbook of clinical pathology Veterinary Clinical Pathology by Ahrar Khan www. medicine.ca/physio/ vlab / bloodlab /diff_count.html bloodjournal.org

26. Laboratory Diagnosis of Bacterial Diseases

27. Methods of bacterial identification Microscopic examination. Cultural appearance. Biochemical reactions. Serological identification. Animal inoculation. Molecular methods.

28. Microscopic examination Gram stain: Pneumococci, gonococci, Staphylococci , meningococci Acid fast stain: Mycobacterium tuberculosis, leprae Fluorescence : -direct - immunoflorescence

29. Cultural characters Pigment production: Endopigment (restricted to the colonies): Golden yellow with Staphylococcus aureus . White with Staph. epidermidis . Exopigment (the color diffuses in the surrounding medium): Green exopigment with Pseudomonas aeruginosa .

30. Hemolysis on blood agar: Complete (beta) hemolysis : Staphylococcus aureus and Streptococcus pyogenes . Partial (alpha) hemolysis : Streptococcus viridans and pneumococci . No (gamma) hemolysis : Enterococci .

31. Effect on lactose of MacConkey’s agar: Lactose fermenters : Appear as rose pink colonies. Example: E. coli & klebsiella . Non Lactose fermenters : Appear as pale colonies. Example: salmonella & shigella .

32. Biochemical reactions Sugar fermentation Indol test Urease test Oxidase test Catalase test Coagulase test DNAse test Gelatinase test

33. - Catalase test : Is used to differentiate between staphylococci( catalase + ve ) and streptococci( catalase – ve ). Principle: Catalase enzyme 2 H 2 O 2 2 H 2 O + O 2 Procedure Smear a colony of the organism to a slide Drop H 2 O 2 onto smear Observe

34. Coagulase test is used to differentiate  Staphylococcus aureus  from coagulase -negative staphylococci. fibrinogen fibrin (clot formation) coagulase

35. Oxidase Test All Enterobacteriaceae are oxidase - negative . This test is used to differentiate enterobacteriaceae from Pseudomonas which is oxidase positive .

36. Motility test to determine whether a bacterium is motile. Non-motile organisms which lack flagella, are usually going to form a single line of growth that does not spread into the surrounding area. While a motile bacterium will grow and make a hazy zone around the stab line.

37. Analytical profile index (API):

38. Automated bacterial identification systems: Principle: Examples: Vitek system These systems identify the organism and its antibiotic sensitivity by detecting color changes or turbidity in special plastic cards inoculated with the organism. Such cards are composed of tiny wells that contain substrates for detection of biochemical reactions and antibiotic sensitivity. Once the card has been inoculated and placed in the instrument, it will automatically perform all readings. Results are available within 4-6 hours.

39. Vitek system Vitek card

40. Serological identification Antigen detection e.g. latex agglutination Antibody detection e. g. agglutination tests, complement fixation tests, indirect immunofluorescence

41. Animal inoculation The use of laboratory animals (mice, guinea pigs, rabbits) is now limited due to the advancement in medical microbiological techniques. 1. For growing the organisms that do not grow on culture such as M. lepra bacilli . 2. To determine the virulence factor of an organism. For example if injection of diphtheria in a guinea pig caused its death, this means that the organism is toxigenic .

42. Laboratory Diagnosis of Virus Diseases

43. Four methods to diagnosis of viral infection: 1- Direct detection of virus/physical method 2- Indirect detection / biological method (virus isolation) 3- Immunology (detection of antibody) 4- Detection of viral nucleic acid (molecular techniques)

44. 1. physical methods (microscope). Electron microscopy (EM) a technique used for obtaining high resolution images of biological A n d n o n-b i o l o g i c al sp ec i m ens u p t o t w o m il li o n t i mes t h an l ig h t microscope. Scanning Electron Microscope (SEM) In a scanning electron microscope or SEM, a beam of electrons scans the surface of a sample (Figure below). The electrons interact with the material in a way that triggers the emission of secondary electrons. These secondary electrons are captured by a detector, which forms an image of the surface of the sample.

45.  SEM  TEM  Belhaouari , 2020

46. 2- Indirect detection / biological method (virus isolation) * virus isolation : There are three methods are used for isolation of viruses: - a. Animals inoculation. b. Chick embryo.

47. c. Cell culture.

48. 3-Immunological techniques: (detection of antibody) Immunological techniques are used to diagnose virus diseases. This is done by demonstrating an antigen-antibody reaction of virus infection. There are several serological and immunological methods and these methods include: ELISA Complement fixation (CF) Latex Agglutination Haemagglutination Assay (HA) and Haemagglutination Inhibition (HI) Virus neutralization Immunofluorescence techniques (IF) Agar Gel diffusion and Agar Gel Precipitation

49. Rapid test

50. 4- Molecular Methods Molecular methods do not rely on the presence of a live virus like virus isolation procedures. These methods detect a piece of the viral genome, making them more sensitive for the detection of viruses. The technique that has probably had the greatest impact on the field of a- Polymerase chain reaction (PCR, identification of DNA) b- Reverse transcriptase polymerase chain reaction (RT-PCR, identification of RNA)virology are