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Interpretation of cbc 2

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continuation from first part with parasitology in peripheral smear and WBC Studies

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Interpretation of cbc 2

  1. 1. Interpretation of CBC contt… DR. N. BAJAJ
  2. 2. Hemoparasites: in peripheral smears
  3. 3. Malaria  Giemsa stain are used, identifies species and life cycle stages  Paresitemia is quantifiable  Threshold of detection thin film: 100 parasites/ L, thick film: 2-20 parasite/L Thick film Thin film • Lysed RBCs • Larger volume • 0.25microliter / 100 fields blood element more concentrated • Good screening for positive or negative parasitemia and parasite density difficult to diagnose species • Fixed RBCs • Single layer • Smaller volume • 0.005 microliter blood required • Good species differentiation • Requires more time to ready A. Peripheral smear
  4. 4. Appearance of P.falciparum in the blood films Ring or trophozoite  Many cells infected – same with more than one parasite  Red cell size unaltered  Parasite is often attatch to the margin of the host cell: called as accole form (arrow) Schizont  Very rarely seem except in cerebral malaria  A single brown pigment dot along with 18-32 merozoites Gamatocyte  Sickle shape “cresent”  Matuer gametocyte is about 1.5 times larger than RBC harbouring it  Microgamatocyte: Broader, shorter, blunt ends. Cytoplasm light blue  Macrogamatocytes: Longer, narrower, pointed ends. Cytoplasm deep blue
  5. 5. Appearance of P. vivex in film Ring or trophozoite  Many cells infected – same with more than one parasite  Unoccupied portion by parasite shows a dotted or stripped appearance “Schuffner’s dot” Schizont  Represent the full grown trophozoite  Contain 12-24 merozoits  Arranged in the form of rosette with yellow brown pigment at the center Gamatocyte  Certain schizont get modified and result in sexual forms. Merozoite arising from single schizont are either all males or females  Microgamatocyte: Spherical. Cytoplasm light blue  Macrogamatocytes: spherical. Cytoplasm deep blue
  6. 6. B. Flouroscien Microscopy  Dyes detect RNA and DNA contents of parasite  Nucleic material not normally seen in RBCs without parasitemia C. Quantative Buffy Coat (QBC)(Becton Dickenson)  Flouroscien microscopy after centrifuge  More sensitive than light microscopy  Useful for screening large number of samples  Quick and saves times
  7. 7. D. Malarial serology – antibody detection  Antibodies to asexual parasite appears some days to invasion of RBCs and may persists for months  Positive test indicate past infection  Not useful for treatment decisions  Valuable epidemiological tools E. Malarial serology – antigen detection  Immunological assey to detect specific antigens  Monoclonal and polyclonal antibodies used in antigen capture tests  Specific species and pan specific antibody  Cross reactivity with other immunological conditions
  8. 8. Malarial antigen detection - RDTs Feature PfHRP 2 PLDH Principle Use monoclonal antibodies Detect HRP of Pf Use monoclonal and polyclonal antibodies Advantage • Threshold for parasite detection ad low as 10 parasites/ microliter • Does not react with other species • Threshold for parasite detection ≥ 100 parasite/ microliter • Can detect all parasite Disadvantages • Sensitivity and specificity decreases as low as 10 parasite / microL • May remain positive upto 14 days posttreatment • Cannot detect mixed infection • Sensitivity and specificity decreases < 100 parasite/ microliter Sensitivity and specificity • Sensitivity 94-100% • Specificity 88-100% • Sensitivity pf 88-98%, pv 84-94% • Specificity pf 93-99%,pv 99-100%
  9. 9. G. Real time PCR  Molecular technique to identify parasite genetic materials  Threshold for detection 1.1 parasite/µL if whole blood is used, if filter paper used it is 2 parasite/ parasite/µL  Species diagnosis present F. Polymerase chain reaction
  10. 10. H. Automation based malaria technique  Hematological parameter and their different combination predict presence of malaria  Low platelet count as strongest predictors, variable MPW, normal to low Plateletcrit and PDW  TLC can be increase or decrease. Leukopenia more seen  Normocytic normochromic anemia, low Hb, decrease RBC count raised ESR, low MCV, MCH, MCHC  Intracellular pigments can also be detected
  11. 11. Filaria  Lymphatic – wucheria, Brugia  Subcutaneous: Loa loa  Sereous: Mansonella  Sample collection between 10 pm to 4 am  Appearance of microfilaria  Measurement 290x7micron  Covered with sheath  Nuclei present all over but not at the tip of tail  Neclei are brolen at different point serving as lamdmark for identification
  12. 12. Babesia  Parasite are intracellular amastigote form. Essential parasite of RE System  Amastigote form are seen in monocytes, less commonly neutrophils Leishmania  Infect mice. Transmitted in between host by ticks  Infected humans may be asymptomatic, but in asplenic host fever, myalgia, haemolysis can be seen  Maltase cross seen in PS
  13. 13. Trypanosoma Cruzi- Chagas disease  Trypanomastigote in peripheral blood  Amastigote in striated muscles
  14. 14. White blood cells
  15. 15. White blood cells  The term leuckocyte is derived from Greek word leukos = white and cyte = cells.  However blood plasma appears green if there is large amounts of neutrophils in the sample, due to haem containing enzyme Myleoperoxidase Normal counts Age Count Birth 4-40 x 109/L 4 years 5-15 x 109/L Adult 4-11 x 109/L
  16. 16. Types Granulocyte (polymorphonuclear) Agranulocyte (mononuclear) Contain membrane bound granules, which stains differently with stains Apparently absent granules, but contain non specific azurophilic granules E.g. Neutrophils Basophil Esionophil E.g. Lymphocyte Monocyte Macrophage
  17. 17. Leukocytosis  High count usually indicate 1. Increase production of WBC to fight infection 2. Reaction to drug that enhance WBC production 3. Disease of marrow, causing high production of WBC 4. An immune system disorder that increase WBC
  18. 18. Leukocytosis Leukopenia Acute and chronic infections Polycythemia vera Rheumatoid arthritis Drugs Allergy ALL AML CLL CML Hairy cell leukamia Smoking Stress Tissue damage such as burns Lymphoma Spillage Measles Myleofibrosis Chemotherapy or radiotherapy Sepsis Typhoid Malaria Tuberculosis Dengue Folate deficiency Drugs like antipsychotic Aplastic anemia HIV and AIDS SLE Hodkins lymphoma Rickettsial infections Pseudo-leucopenia Seen during the onset of infections due to marginated WBC
  19. 19. Band cells  Usually constitute <5-10% of white blood cells  An increase in number of band cell and other immature neutrophils is called a “ shift to left” can be seen in  Severe infections, sepsis  Non infectious inflammatory disease  Pregnancy
  20. 20. Causes of increased neutrophil: ANC >75000/cumm 1. Physiologic increase (Demargination) • Release of cell in marginal pool • Stress leukocytosis • Exercise, Seizure • Anxiety, Epinephrine 2. Acute infections 3. Tissue injury and inflammation • Collagen vascular disease • Hypersensitivity • Burns 4. Myeloproliferative disorders: myeloid leukemia, polycythemia vera 5. Medications: Corticosteroid, lithium 6. Misc.: Sickle cell anemia, acute hemorrhage Causes of Neutopenia 1. Decrease or ineffective production • Aplastic anemia • Drug • Deficiency – vitamin B12, Folic acid • Myelodysplastic syndrome • Inherited disorder – Kostamann synd. 2. Increased removal from circulation • Immunological – SLE, Drugs • Hypersplenism
  21. 21. Hematological Scoring System (HSS): Neonatal Sepsis  HSS can be very useful to differentiate the infected from non-infected infants  It has high sensitivity and specificity  An immature to total neutrophil ratio [I:T] along with degenerative changes > immature to mature [I:M] is the most sensitive indicator of sepsis in infant  Immature include: promyleocyte, myleocyte, metamyleocyte and band cells  Degenerative changes: vacuolization, toxic granules and Dohle bodies.  Confirmation by Blood Culture
  22. 22. Hematological Scoring System (HSS) Criteria Abnormality Score WBC <5000/microL >25000 at birth >30000-(12-24h) >21000 day 2 onwards 1 1 1 1 Total PMN count No mature PMN seen Increase/ destruction 2 1 Immature PMN count Increased 1 Immature: total PMN ratio Increased 1 Immature: mature PMN ratio >0.3 1 Degenerative changes in PMN Toxic granules/ Vacuoles 1 Platelet count <150000 microL 1 Score Interpretation <2 Sepsis is unlikely 3-4 Sepsis is possible >5 Sepsis or infrction is very likely
  23. 23. Cytokinin Myeloid precursor in bone marrow Cell Cell is altered White cells more readly exits marrow Increase phagocytic activity Cytoplasmic inclusions may appear Enhanced enzyme production and packing resulting in large granules Toxic vacoulisation Left shift Dohle bodies Increase phagocytic activity Cytoplasmic inclusions may appear Left shift Toxic vacoulisation
  24. 24. Toxic granulation and vacuolization  Indicate the presence of increased granules that are larger and more basophilic in normal  Seen in  Severe infection  Aplastic anemia  Burns  Malignancy  Treatment with CSF  Pregnancy
  25. 25. Dohle bodies  Composed of rough endoplasmic reticulum and glycogen granules  Small blue grey inclusion seen in neutrophil usually in periphery  Seen in  Infections  Inflammatory disorders  Pernicious anemia  Myeloproliferative disorders  Myelodysplastic disorders  Cancer chemothrapy
  26. 26. Hypersegmentation  Exists when > 5% of neutrophils have 5 or more lobes  Seen in folate and vitamin B12 deficiency  Myeloproliferative disorders  Myelodysplastic disorders
  27. 27. Pelger Huet anamoly  70-90% neutrophils have hypolobulated, rounded nuclei, with condense chromatine  A thin strand connect the lobes giving rise to pince-nez (spectacle) shape, or a larger bridge give rise to peanut shape.  Heridetery hypolobulation has no significance  Acquired (Pseudo Pelger Huet) anamoly, common in myelodysplastic and myeloproliferative disorders
  28. 28. Auer Rods  Seen in myeloid blast of acute leukemia  They are fused lysosomes and contain lysosomal enzyme and large crystalline inclusions, seen in the cytoplasm of leukemic blast  They are virtually pathognomic of myeloid leukemia
  29. 29. Leukamoid reactions  Leukamoid reaction is a haematological disorder that simulates leukemia due to high WBC counts and presence of some immature leukocytes. In leukamoid reaction the cells are not clonally derived.  Persistant neutrophilia with cell count of >30000-50000/microL is called myeloid leukamoid reaction  Leukamoid alkaline phosphate score (LAP- score) can differentiate leukamoid reaction from CML. LAP score is raised in leukamoid reaction whereas decreased in CML
  30. 30. Some causes of leukamoid reaction Causes Myelocytic Lymphocytic Monocytic Infections Endocarditis Pneumonia septicemia Leptospirosis etc. Infectious mononucleosis Pertusis Varicella Tuberculosis Tuberculosis Toxic conditions Burns Poisoning -mercury Eclampsia Neoplasia Ca Colon Embryonal carcinoma of kidney Carcinoma of stomach Carcinoma of breast Others Acute haemorrhage Acute haemolysis Dermatitis herpitiformis
  31. 31. Eosinophils  Cells having large dinstintive red orange specific granules in cytoplasm, which contain histamine and other substances  Lives 6-12 hours in circulation, migrate into tissues  Normal range: 1-4% of total WBCs  Absolute count: 12-500cells/ microliter  Diurnal variation – related to cortisol level: lowest in morning, highest in evening
  32. 32. Eosinophilia Eosinopenia Mild 700-1500 • Allergic rhinitis • Extrinsic asthma • Mild drug reaction • immunodeficiency Usually related to increased steroids Cushing syndrome Drugs ACTH, epinephrine, thyroxine Acute bacterial infections Moderate 1500-5000 Parasitic disease Intrinsic asthma Pulmonary Eosinophilia syndrome Marked >5000 Trichinella Hookworm Toxocara canis Severe drug reaction Eosinophilic leukamia
  33. 33. Basophils  Contain large purplish granules, granules obscuring the nucleus  Releases bradykinin, heparin, serotonin, histamine  Mediates allergic reactions  Circulate for few hours(6-12) then migrates into tissue  Range 0.5-2%, absolute count 6-200 /microloiter  Basophilia - causes  Hypothyroidsm  CML  Ulcerative colitis  Polycythemia vera  Uticaria  Chickenpox  Splenectomy
  34. 34. Monocytes  Agranulocytes, contain greyblue granules  Life span 8hrs-30days, migrate in tissue and became macrophage Monocytosis Monocytopenia >700 /mcL or >12% WBC • Viral infections • Tuberculosis • Sub acute bacterial endocarditis • Collagen disease • Chronic inflammation • Stress • Infectious mononucleosis • Sarcoidosis • Autoimmune • SLE • Rheumatoid disease Hairy cell leukemia Aplastic anemia
  35. 35. Lymphocytes Lymphocytosis Lymphocytopenia Infectious mononucleosis Tuberculosis Brucellosis Cytomegalvirus Rubella toxoplasma Hepatitis A,B Wooping cough ALL Burkitt lymphoma CLL Hairycell leukemia Non Hodgkin's lymphoma X- linked lymphoproliferative disorders Viral infections HIV SARS Marrow suppression Pancytopenia Drugs: Vinblastin, Doxorubicin, Chromphenicol
  36. 36. Platelets  Thrombopoiesis take place in bone marrow  1 megakaryocyte produce 4000 platelets  Normal platelet are about 1.3 micron, blue grey, contain fine, purple to pink granules  Red cell to platelet ratio : 10- 40:1  Life span 9-12 days  Range : 1.5-4.5 lakhs/microL
  37. 37. Thrombocytopenia  Grade 1- counts is between 75,000 -150,000  Grade 2- counts is 50,000 < 75,000  Grade 3 – 25,000 to < 50,000  Grade 4 - < 25,000
  38. 38. Thrombocytopenia Decrease production Increase destruction Abnormal distribution • TAR syndrome • Amegakaryocytic thrombocytopenia • Aplastic anemia • Myelodysplatic synd • Bome marrow hypoplasia or infiltration • Ineffective thrombopoisis due to folate deficiency • Heridietery • May Hegglin anamoly • Wiskott Aldrich Syndome • Immune mediated • SLE • ITP • Drugs like heparin • HIV • Posttransfudion purpura • Non immune • Severe bleeding • DIC • Vasculitis • vWD • TTP • HUS Hypersplenism Dilutional, due to massive transfusion
  39. 39. Pseudo- thrombocytopenia (artifactual) A. EDTA induced platelet agglutination  This is invitro phenomenon due to presence if auto antibodies against a crypt antigen on the GP IIb/ IIIa receptor, when calcium is chelated by EDTA, the GPIIb/IIIa get exposed and causes agglutination of platelets  Occurs in 1% of hospitalized patients  No evidence of abnormal haemostasis  Confirmed by sampling on citrated blood B. Platelet satellitsm: platelet rossete formed around the neutrophil or any other cells. These satellite platelets are not counted by counter. It is caused by EDTA dependent antiplated and antineutrophil IgG antibodies. It is not associated with any disease
  40. 40. C. Cold agglutinin: temperature dependent phenomenon. Sample has to be warm to 37 degree C to get accurate platelet count D. Giant platelet or Megakaryocyte: platelet larger than 36fl is counted as red cell in counter, resulting in low platelet count  Mean platelet volume is increase in giant platelets  Young platelets are usually larger  Causes of large platelets include:  Hereditary – Bernard Soulier Syndrome, Benign Mediterranean macrothrombocytopenia  Acquired – immune thrombocytopenia purpura  Myeloproliferative syndrome  Myleodysplasia  DIC  TTP  Partially clotted specimen: some platelets get consumed
  41. 41. Thromboasthenia: Platelets with normal count but abnormal function, leading to episodes of bleeding (A) Inherited : (B) Acquired 1. Aggregation defect: Glanzmann thrombosthenia, congenital afibrinogenemia 2. Platelet adhesion defect: Bernard Soulier syndrome, vBD 3. Signaling pathway defect: defect in calcium mobilization, thromboxane synthetase deficiency,cyclooxygenase and lipoxygenase deficiency 4. Agonist receptor defect: thromboxane receptor deficiency 5. Secretion defects: Chediak Higasi synd, storage pool disease, Wiskott Aldrich synd, Grey platelet syndrome 1. Essential thrombocytopenia 2. Uremia 3. Antiplatelet antibodies 4. Myeloproliferative disorders 5. Polycythemia vera 6. CML 7. Acute leukemia 8. Myleodysplastic syndrome 9. vWD 10.Liver disorders
  42. 42. Thrombocytosis Myleoproliferative disorders Transfer from extravascular pool Thrombocytosis secondary to Essential thrombocytosis Idiopathic myleofibrosis Polycythemia vera Chronic granulomatous leukemia Splenectomy Exercise Epinephrine Iron deficiency Infections Hemolysis Malignancy Acute blood loss
  43. 43. Mean platelet volume - MPV  Measurment that describe the average size of the platelet in the blood.  It is indicator weather bonemarrow is manufacturing platelets normally or there is some kind of production pressure  MPV has inverse relation with platelet count  change in mean platelet volume without any change in platelet count may be early indicator of bone marrow problem  Platelet are considered large when 49-8 micron diameter and giant when equals RBCs Normal range – 7.4-10.4fL
  44. 44. Increase MPV (megathrombocytes) Decrease MPV ITP TTP Bernard Soulier synd May Hagglin disease Sepsis - recovery phase Heart valve prosthesis Myelodysplasia Sickle cell anemia Hyperthyroidsm Aplastic anemia Wiskott Aldrich syndrome TAR synd Storage pool disease Megaloblastic anemia Hypersplenism Note: in general platelets are large when thrombocytopenia results from increased destruction and small with disorders of diminished production. If platelet count is low and MPH is high the risk of bleeding is comparatively less as larger platelets have multifold better hemostatic capacity than normal size platelet.
  45. 45. Platelet distribution width (PDW)  Compares uniformity and heterogeneity of platelet size; as RDW  Increased in  Essential thrombocytopenia  Aplastic anemia  Megaloblastic anemia  CML  Chemotherapy  Fragmented RBCs  PDW is a relative good tool to distinguishessential thrombocythemia (PDW increase) from reactive thrombocytosis (PDW normal)
  46. 46. Plateletcrit  It is the volume percentage that platelets match on total blood volume of blood, and it is directly related to the total volume of the platelets and MPV  Normal Range 0.110-0.280
  47. 47. Peripheral smear in thrombocytopenia RBC lineage • Schistocytes Microangiopathic haemolytic anemia, DIC, HUS • Malaria parasite Thrombocytopenia, pf • Spherocytes AIHA+thrombocytopenia (Evan Syndrome) • Normoblast and polychromasia HELLP • Autoagglutination Cold antibodies WBC lineage Increase polymorphs Infection/ septicima Toxic granules band cells Septicimia Precursor cells, blast cells Leukaemia Dysplastic cells MDS Platelet lineage Giant platelets ITP,BSS, May Heglin, grey platelet synd, montreal platel,sebstian syndrome Scattered platelet in direct smear Glanzmann synd
  48. 48. Manual vs Automation hematology  Manual with Neubauer chamber are used mainly used where there is economic considerations and non availability of automation  Disadvantages of manual counting  Cell identification:  mostly between lymphocyte, monocytes, band cells  Segmented form and abnormal cells  Lymphocytes may be over estimated and monocytes may be underestimated  Cell distribution error : increased cell concentration along edges and also bigger cells  Statistical sampling errors
  49. 49. Automated counters provides a 3 or 5 or 7 part differential count 3 part differentiation 5 part differentiation 7 part differentiation 1. Granulocytes or larger cells 2. Lymphocyte or smaller cells 3. Monocytes or mid cell population 1. Neutrophils 2. Eisonophils 3. Basophils 4. Lymphocytes 5. Monocytes 6. A sixth category “large” unstained cells, include cells larger than normal and lacks peroxidase activity- atypical lymphocytes and other abnormal cells Include 5 part • Large immature cells- blast and immature granulocytes • Atypical lymphocytes
  50. 50. Cell counter – basic principle  Inventor - Wallace Coulter  Electrical impedence principle of cell counting: The cell size are counted by detecting and measuring changes in the electrical resistance when a particle passes through a small aperture.  Mathematically V=RxC V-voltage, C – current, R= resistance
  51. 51.  The electrical system : circuitry, sequence controls, transformers  The hydraulic system : aspirating unit, dispenser, diluents, mixing chambers, flow cells, aperture bathes and haemoglobinometre  Pneumatic system : vacuum and pressure devices  Computer system Cell counter – basic components
  52. 52. Radiofrequency principle of cell counter  This employ high voltage electromagnetic currents, which can estimate the cell size based on cellular density and nuclear volume  It measures the conductivity and the conductivity is altered by nuclear to cytoplasmic ratio, nuclear density, granulation. VCS principle of cell counter  VCS= volume, conductivity, scatter  Direct current – measures the size of the leukocytes based upon its volume  Conductivity – HF radiowaves measures conductivity of the cells  Scatter – laser light beam evaluates the surface feature, structure, shape, granularity and reflectivity
  53. 53. Coincidental or Recirculation errors  If more than 1 cell passes through the counting aperture at the same time and is counted as one cell, this is called coincidental error
  54. 54. Advantages of automated cell counters  No inter-observer variability  No slide distribution errors  Eliminate statistical error  Many parameter are available e.g. RDW, histogram  More efficient and time effective  High level of precision and accuracy
  55. 55. Thanks and give blood

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