Peripheral blood smear [autosaved]

peripheral blood smear by Dr Akansha anu

1. PERIPHERAL BLOOD SMEAR dr. akansha anu Guide dr. Vaishali kotasthane

2. INTRODUCTION Peripheral smear is the most important investigation in haematology. It provides information about red cells –their number, shape, size and variations in morphology. It helps in diagnosis of different types of anaemias and other haematological disorders. DLC is very important diagnosis of various haematologic and non haematologic diseases. Assessment of platelet number, their aggregates and morphology is helpful in diagnosis of various bleeding disorders. Smear evaluation is a check on the value obtained from automated cell counters. For evaluation of PS it is important to have a nicely made and stained PS.

3. Role of peripheral blood examination. Evaluation of anaemia. Evaluation of thrombocytopenia/ thrombocytosis. Identification of abnormal cells. Inclusion like basophilic stippling, Howell-jolly bodies. Infection like malaria, microfilaria etc.

4. Collection of blood Advantages Many smears can be done in just a single draw. Immediate preparation of smear is not necessary. Disadvantages Platelets satellitosis causes pseudothromocytopenia and pseudoeukocytosis. Causes :platelet specific auto antibodies that react best at room temperature.

5. Anticoagulants EDTA – Most commonly used anticoagulants (CBC, Hb, TLC, DLC, PLATELET COUNT, RBC COUNT) Sodium citrate – ESR (WESTERGREN METHOD) Double oxalate- used in coagulation studies Heparin – used for coagulation, for red cell enzyme studies like G6PD and pk deficiency Sodium fluoride- estimation of blood glucose

6. Color coded tubes

7. EDTA Collected in lavender (purple) topped tubes Contain disodium or trisodium ethylenediaminetetraacetic (EDTA) anticouglants the blood by chelating the calcium that is essential for coagulation High quality of blood films can be made within 2-3 hrs of drawing Blood films from EDTA tubes that remain at room temperature for more than 5 hrs often have unacceptable blood cell artifacts Echinocyte red blood cells Spherocytes Degenerated leukocytes Vacuolated neutrophils

8. Characteristic of good smear Good smear is tongue shaped with a smooth tail. Does not cover the entire area of the slide. Has both thick and thin areas with gradual transition. Does not contain any lines or holes.

9. Preparation of smear There are three types of blood smear The wedge smear The cover glass smear The spun smear There are two additional types of blood smear used for specific purposes Buffy coat smear Thick blood smeras for blood parasites

10. Wedge technique Easiest to make Most convienient and most commonoly used technique EQUIPMENT Spreaders Clean slides Blood capillary tube or micropipette 10ul

11. Place a drop of blood, about 2-3 mm in diameter approximately 1cm from one end of slides.

12. Place the slide on a flat surface, and hold the other end between your left thumb and forefinger. With your right hand, place the smooth clean edge of second (spreaders) slide on specimen slide, just in front of blood drop. Hold the spreaders slide at a 30 degree – 45 degree angle, and draw it back against the drop of blood. PRECAUTIONS Too large drop =too thick smear Too small drop=too thin smear

13. Allow the blood to spread almost to the edges of the slide PRECAUTIONS . Ensure that the whole drop of blood is picked up and spread ANGLE CORRECTION : High Hct: angle should be lowered Low Hct: angle should be raised

14. Cover slip technique Rarely used ADVANTAGE- excellent leucocyte distribution. DISADVANTAGES-labelling, transport, staning and storage is a problem. TECHNIQUE- A drop of blood is placed on top of 1 coverslip. Another coverslip is placed over the other allowing the blood to spread. One is pulled over the other to create 1 thin smears. Mounted on a 3x1 inch glass slide.

15. Automated slide making and staning . Perfoms a CBC for specimen. Dependent on the hemocrait reading, the system adjusts. Size of the drop of blood used and Angle and spread of the spreaders slide in making a wedge preparation. After each blood flim is prepared, the spreadrers slide is automatically cleaned.

16. Automated slide making and stanning. Films be produced approximately every 30 seconds. Name, number, and date for the specimen is printed on the slide. The slide is dried, loaded into a cassette,and moved to the stanning position, where a stain and then buffer and rinse are added designated times. When stanning is complete, the slide is moved to a dry position, then to a collection area where it can be picked up for microscopic evaluation.

17. Stains for blood smear Romanowsky stains are universally employed for stanning of blood smears It combines of methylene blue and eosin Basic dye Has affinity for acidic component of the cell i.e. nucleus and eosin has affinity for basic component I;e basic Various stains for Romanowsky are; 1.Leishman’s stain 2. giemsa stain 3 wright stain 4 field stain 5 jenner stain 6 JSB stain

18. Staning of thin blood smear Leishman’s stain Preparation Dissolve 0.2 g of powdered LS dye in 100ml of acetone free methyl alcohol Warm it to 50 degree c for half n hr with occasional shaking Cool it and filter it Procedure Pour LS dropwise on slide and wait for 2 mins (allows fixation) Add double the quality of buffered water over the slide Wash in water for 1 -2 mins Dry in air and examine under oil immersion

19. Giemsa stain Prepartion Mix 0.15 g of giemsa powder in 12.5 ml of glycerine and 12.5 ml of methyl alcohol Before use dissolve one volume of stock solution in nine volumes of buffered water (dilution 1:9) Procedure pour diluted stain over slide or immense blood smear in staning trough Wait for 15-60 mins Wash in water Dry it and examine under oil immersion

20. AUTOMATED SLIDE STAINERS It takes about 5-10 mins to stain a batch of smears. Slides are just automatically dipped in the stain in the buffer and a series of rinses. DISADVANTAGES Stanning process has begun, no stat slides can be added in the batch. Aqueous solutions of stains are stable only for 3-6 hours.

21. Rapid stanning method-field’s stain Advantage fast, convenient and takes about 1 minute. Cost effective. Components. Methanol Solution b contains eosin Solution a contains methylene blue

22. MICROSCOPIC OVERVIEW

23. CAUSES AND CORRECTIONS Too acidic stain. Insufficient staining time. Prolonged buffering or wasting Old stain Correction Lengthen staning time Check stain and buffer ph Shorten buffering or wash time

24. Too alkaline stain Thick blood sugar Prolonged standing Insufficient washing Alkaline ph. of stain components Corrections Check ph. Shorten stain time Prolonged buffering time

25. Features of a well-stained PBS Microscopically- color should be pink to purple. Microscopically- RBC orange to salmon pink WBS- nuclei is purple to blue Cytoplasm is purple to pinl Granules is iliac to violet Eosinophil- granules orange Basophill - granules dark blue to black.

26. MORPHOLOGIC CHANGE DUE TO AREA OF SMEAR Thin area- spherocytes which are really spheroidocytes or flayttened red cells. True spherocytes will be found in other good areas of smear. Thick area-rouleaux which is normal in such areas. Confirm by examining thin areas. If true rouleaux's two three RBCs will stick together in a stack of coins fashion.

27. 10x objective Assess overall quality of the smear i.e feathery edge, quality of color, distribution of cells and the lateral edges can be checked for WBC distribution. Snow plow effect ; more than 4x cells per field on the feathery edge; reject Fibrin stands; reject

28. TOTAL LEUCOCYTE COUNT 40 X OBJECTIVE Use dry without oil Choose a portion of the peripheral smear where there is only slight overlapping of RBCs Count 10 fields take the total number of white cells and divide by 10. To do a wbc estimate by taking the average number of white blood cells and multiplying by 2000 Normal leucocyte count ranges from 4000 to 11000/ul

29. Observe one field and record the number of WBC according to the different type then turn to another field in snake like direction.

30. Manual differential counts These counts are done in same area as WBCs and platelet estimates with the red cells barely touching This takes place under x 100 (oil) using the zigzag method Count 100 WBCs Expressed as percentage Absolute number of cells/ul = %of cell type in differential x white cell count

31. NUCLEATED RED BLOOD CELLS If 10 or more nucleated RBCs are seen correct the TLC Corrected WBC count = WBC x 100 / ( nRBC +100) EXAMPLE If WBC =5000 and 10 nRBCs have been counted Then 5000 x 100/110 =4545 Then corrected white count is 4545

32. Do not count Disintegrating cells Eosinophil with no cytoplasmic membrane and with scattered granules Smudge cells Pyknotic cells

33. RBC MORPHOLOGY Scan under using x 100 (Oil immersion) Observe 10 fields Red cells are observed for – size, shape, haemoglobin content, inclusions

34. RBC RBCs are circular, homogenous disc nearly of uniform size (7-8 um) Deep pink cytoplasm with central pallor <1/3 rd )

35. HYPOCHROMIA Decrease in haemoglobin content of RBC Increase in central pallor(1/3) Decrease in MCH and MCHC Seen in various anaemias

36. Dimorphic anaemia Presence of anisocytosis and anis chromia in same film Seen in- coexistence of iron deficiency and megaloblastic anaemia. Sideroblastic anaemia Some weeks after iron therapy for iron deficiency anaemia. Hypochromic anaemia after transfusion with normal cells.

37. Variation in size

38. MICROCYTES Size of RBCs are reduced (<80fl) Seen in – iron deficiency anaemia thalassemia anaemia of chronic disease sideroblastic anaemia

39. Macrocytes When MCH of RBC is increased (>100fl) Seen in – vit b12 and folate deficiency alcoholism hepatic disease haemolytic states hypothyroidism

40. Shape Variations in shape is called poikilocytoses Types Elliptocytes Spherocytes Target cells Schistocytes Acanthocytes Karyocytes Echinocytes

41. ELLIPTOCYTES Elipitical in shapes Most abundant in hereditary elliptocytes Seen in- iron deficiency anemia megaloblastic anemia

42. Acanthocytes Thorny projections on red cell membrane Few irregular non uniform Seen in – hypothyroidism liver disease McLeod phenotype

43. Echinocytes (Burr cells) Numerous short regular projection Commonly occur as an artifact during preparation Renal disease Liver disease hyperlipidaemia

44. LEPTOCYTES Thin red cells with large unstained central area Also known as pessary cells Seen in – iron deficiency anaemia thalassemia

45. Somatotypes Red cells with central biconcave area appears slit like in dried film Seen in- liver disease hereditary alcoholism myelodysplastic syndromes

46. Sickle cell Cells are sickle (crescent) shape Present in film of patient with homozygosity for HbS

47. Tear drop cells Also called dacrocytosis Seen in- beta thalassemia post splenectomy severe iron deficiency

48. Spherocytes Nearly spherical Diameter is smaller than normal Lack central pale area or have smaller, eccentric pale area Seen in- hereditary spherocytes autoimmune haemolytic anaemia physical or chemical injury

49. Target cells Cells in which central round stained area and peripheral rim of cytoplasm Seen in – sickle cell anemia thalassemia major hemoytic anemias postsplenectomy

50. RBC INCLUSION

51. HOWELL- JOLLY BODIES Smooth single large round inclusion which are remnant of nuclear chromatin. Seen in- megaloblastic anaemia haemolytic anaemia postsplectomy abnormal erythropoiesis

52. BASOPHILIC STIPPLING Presence of irregular basophilic granules with in RBCs which are variable in size Fine stippling seen Coarse stippling- lead and heavy metal poisoning disturbed erythropoiesis megaloblastic anaemia thalassaemia infection liver disease

53. Pappenheimer bodies Smaller than Howell- jolly bodies Composed of haemosiderin Seen in - hyposplenesim myodysplastic syndrome

54. Heinz bodies Purple blue large single or multiple inclusion attach to inner surface of red blood cells Seen in – post splenectomy oxidative stress drugs toxins glutathione synthetase deficiency

55. Cabot ring These are ring shaped figure of eight or loop shaped Observed in – megaloblastic anemia pernicious anemia lead poisoning

56. Rouleaux formation Alignment of red cells one upon another so that resemble stack of coins Occurs in – multiple myeloma chronic inflammatory disease

57. Agglutination It is more irregular and round clumping than liner Rolex Seen in – anti RBC antibody autoimmune haemolytic anaemia macroglobulinemia

58. WBCs in PBS GRANULOCYTES Neutrophils Eosinophils Basophils Agranulocytes Lymphocytes monocytes

59. Polymorphonuclear neutrophills The terminal stage of development measuring 12- 14 um in diameter Characterised by a lobulated nucleus Two to five lobes of clumped chromatin The cytoplasm contains fine azurophilic granules

60. Hypersegmented neutrophils Presence of even a single neutrophils with six or more lobes Seen in – megaloblastic anaemia uraemia

61. Eosinophils They are slightly larger than a segmented neutrophil measuring 12-15 um Two nuclear lobes are spectacle in shape The cytoplasm has pale hue and has numerous dense orange red colour

62. Monocytes Monocytes are 10-11um The nucleus is large and oval The nuclear chromatin are delicate The cytoplasm is abundant, is grey or light blue grey in colour The granules resemble fine dust and give bluish cytoplasm a ground glass appearance

63. Monocities Chronic infections and inflammatory conditions such as : Malaria Typhoid Kala-azar Bacterial endocarditis Crohn’s disease Infectious mononucleosis tuberculosis Haematolymphoid malignancies Acute myelomonocytic leukaemia(AML M4) acute monocytic leukaemia (AML M5) Myeloproliferative neoplasm Myelodysplastic syndrome Chronic myelomonocytic leukaemia

64. Lymphocytes Small lymphocytes Measuring 9-12um Smaller than granulocytes Cytoplasm is in the from of thin rim around the nucleus Round and slightly intended nucleus

65. Large lymphocytes Measuring 12 -15um Round in outline Nucleus is round and slightly indented With clumped chromatin Cytoplasm is more abundant than lymphocytes and pale blue in colour

66. Granules Seen in Bacterial infections Burn Administration of G-CSF GM –CSF

67. Dohle bodies Small round or oval pale blue grey structure Found at periphery of neutrophils Contains ribosomes and endoplasmic reticulum Seen in Bacterial infections Inflammation Pregnancy Administration of G-CSF

68. Vacuoles in neutrophils In fresh blood smear vacuoles seen in severe sepsis Indicative of phagocytosis

69. Alder- reilly anomaly Commonly seen in hurler’s and hunter’s syndrome Granules are large discrete stain deep red Neutophils function is normal

70. May hegglin anomaly Autosomal dominant inheritance Triad of thrombocytopaenia giant platelets and dohle’s bodies MYH -9 gene

71. Chediak-higashi syndrome Rare autosomal recessive disease Immune deficiency Poor resistance to bacterial infections Bleeding tendencies Multiple neurological abnormalities

72. Pelger-huet cells Benign inherited condition Neutrophil nuclei fail to segment properly

73. Platelets size 1-3 um Normal count 1.5 to 4.5 lac/cmm Non nucleated derived from cytoplasmic fragments of megakaryocytes Have an irregular outline and fine purple red granules

74. Thrombocytopenia

75. Thrombocytosis Essential thrombocytopenia CML Reactive thrombocytosis – post infection iron deficiency inflammation collagen vascular disease

76. Platelet morphology – giant platelets Platelets seem to be size of RBCs Seen in Alport syndrome Storage disorders Bernard syndromes

77. Hemoparasites Malaria Microscopic examination of peripheral blood film is the gold standard for diagnosis Number of parasitized RBCs seen in 10,000 RBCs (in 100x objective) is calculated Approximate number of parasites is roughly assessed assuming 1ul of blood contains 5x10’6 RBCs Blood film evaluation Thin film examination Thick blood film evaluation Malaria antigen detection test Molecular method Serology Loop mediated isothermal amplification test

78. Plasmodium falciparum Infected RBCs are of normal size with one or multiple rings Gametocytes have characteristic banana shape Few Maurer's clefts may be seen

79. Plasmodium vivax Infected RBCs are enlarged and deform Parasites infect the reticulocytes which demonstrate ring form, schizonts with dots and gametocytes Gametocytes are large and round to oval with eccentrically placed chromatin

80. Plasmodium ovale Infected erythrocytes Moderately enlarged Oval in shape Show red granules like schuffenr’s dots Merozoites have daisy head diatribution Gametocytes are small ½ to 2/3 rd of red cells

81. Plasmodium malariae Infected RBCs –size normal to decreased Ameboid forms are seen as a band across the red cells The gametocytes are small and round Occupies 1/3 rd to 2/3 rd of red cells Hemozoin is present in liver, spleen, brain

82. Filariasis Causes elephantiasis Many caused by wuchereria bancrofti and brugia malayi Pathology- due to adult worm obstructing lymphactics Includes eosinophilia and elephantiasis of legs and scrotum Diagnosis- PBS membrane filter method immunochromatographic test DNA probe using PCR

83. filariasis Detection of microfilariae in blood in early stages of disese Blood film, knott’s method (concentration of 1 ml of blood) Best 10 pm to 2 am (nocturnal periodicity)

84. trypanosomiasis Transmitted by tse tse fly Causes African sleeping sickness and splenomegaly

85. Babesiosis It is a tick borne disease caused by protozoan caused by parasite babesia Vector is tick Cause malaria like sickness In blood organism recognised as tiny multiple rings in red cells

86. Toxoplasmosis Caused by toxoplasma gondii Infection from cats Immunodeficient cases- involvement of brain, eyes, muscle, heart, lungs Rarely trophozoites are seen in peripheral blood.

87. Thank you