Interpretation of cbc 2

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

1. Interpretation of CBC contt … Dr. N. Bajaj

2. Hemoparasites : in peripheral smears

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 Peripheral smear

4. Appearance of P.falciparum in the blood films Ring or trophozoit e 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. Appearance of P. vivex in film Ring or trophozoit e 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. 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. 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. 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. 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. 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. 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. 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. Trypanosoma Cruzi - Chagas disease Trypanomastigote in peripheral blood Amastigote in striated muscles

14. White blood cells

15. White blood cells The term leuckocyte is d erived from G reek 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 10 9 /L 4 years 5-15 x 10 9 /L Adult 4-11 x 10 9 /L

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. Leukocytosis High count usually indicate Increase production of WBC to fight infection Reaction to drug that enhance WBC production Disease of marrow, causing high production of WBC An immune system disorder that increase WBC

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. 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. Causes of increased neutrophil: ANC >75000/ cumm Physiologic increase ( Demargination ) Release of cell in marginal pool Stress leukocytosis Exercise, Seizure Anxiety, Epinephrine Acute infections Tissue injury and inflammation Collagen vascular disease Hypersensitivity Burns Myeloproliferative disorders: myeloid leukemia, polycythemia vera Medications: Corticosteroid, lithium Misc.: Sickle cell anemia, acute hemorrhage Causes of Neutopenia Decrease or in effective 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. Hematological Scoring System (HSS): N eonatal 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. 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. 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. 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. 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. Hypersegmentation Exists when > 5% of neutrophils have 5 or more lobes Seen in folate and vitamin B 12 deficiency Myeloproliferative disorders Myelodysplastic disorders

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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. Pseudo- thrombocytopenia ( artifactual ) 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 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. Cold agglutinin: temperature dependent phenomenon. Sample has to be warm to 37 degree C to get accurate platelet count 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. Thromboasthenia : Platelets with normal count but abnormal function, leading to episodes of bleeding (A) Inherited : (B) Acquired Aggregation defect : Glanzmann thrombosthenia , congenital afibrinogenemia Platelet adhesion defect: Bernard Soulier syndrome, vBD Signaling pathway defect : defect in calcium mobilization, thromboxane synthetase deficiency,cyclooxygenase and lipoxygenase deficiency Agonist receptor defect: thromboxane receptor deficiency Secretion defects: Chediak Higasi synd , storage pool disease, Wiskott Aldrich synd , Grey platelet syndrome Essential thrombocytopenia Uremia Antiplatelet antibodies Myeloproliferative disorders Polycythemia vera CML Acute leukemia Myleodysplastic syndrome vWD Liver disorders

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. 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. 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. 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. 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. 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. 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. Automated counters provides a 3 or 5 or 7 part differential count 3 part differentiation 5 part differentiation 7 part differentiation Granulocytes or larger cells Lymphocyte or smaller cells Monocytes or mid cell population Neutrophils Eisonophils Basophils Lymphocytes Monocytes 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. 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. 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. 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. 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. 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. Thanks and give blood