Peripheral blood smear pathology...........

Basic knowledge about pbs

1. PERIPHERAL BLOOD SMEAR and hematological staining techniques Presentation by: Dr. Divya Malviya Guided by: Dr. Surabhi Ruiwale

2. Definition A blood smear is a specimen for microscopic examination prepared by spreading a drop of blood across a glass side followed by staining with one of the Romanowsky’s stains.

3. Uses Evaluation of anemia Evaluation of thrombocytopenia/ thrombocytosis Identification of abnormal cells (blasts / abnormal promyelocyte / atypical lymphoid) Diagnosing hemoparasitic infections To provide direction for further investigations that will help in arriving at correct diagnosis.

4. OBJECTIVES Peripheral Smear Preparation Staining of Peripheral Blood Smear Peripheral Smear Examination

5. PREPARATION OF BLOOD SMEAR Techniques for blood smear preparation: Wedge method ( M/C Used). Cover slip method. Automated slide making and staining.

6. Wedge method 1. Place a drop of blood, about 2-3 mm in diameter approximately 1 cm from one end of slide. 2. Place the slide on a flat surface, and hold the other end between your left thumb and forefinger 3. With your right hand, place the smooth clean edge of a second (spreader) slide on the specimen slide, just in front of the blood drop. 4.Hold the spreader slide at a 30°-45 angle, and draw it back against the drop of blood 5.Allow the blood to spread almost to the edges of the slide. 6.Push the spreader forward with one light, smooth moderate speed. A thin film of blood in the shape of tongue is made. 7. Label one edge with patient name, lab id and date. 8. The slides should be rapidly air dried by waving the slides or using an electrical fan.

7. A well-spread blood smear is: (a) is tongue-shaped with a smooth head , body and tail. (b) does not cover the entire area of the slide, (c) has both thick and thin areas with gradual transition, (d) does not contain any lines or holes. In patients with anemia, a thicker smear can be obtained by increasing the angle and the speed of spreading. In patients with polycythemia , a thinner smear is obtained by decreasing the 'spreader' angle and the speed of spreading.

8. Blood film made on slides. (A) A well-made film. (B) An irregular patchy film on a dusty slide. (C) A film that is too thick. (D) A film that has been spread with inconsistent pressure and using an irregularly edged spreader, resulting in long tails. (E) A film made on a very greasy slide. A B C D E

9. Cover Glass Method Touch a clean cover glass to the top of a small drop of blood without touching the skin (when using capillary blood) and place it down Place another cover glass cross- wise on it so that the corners will appear as an eight-pointed star. The blood will spread out evenly and quickly in a thin layer between the two surfaces. Separate the two cover slips by pulling them in opposite direction Cover glasses should be placed film side up on a clean paper and allowed to dry in the air After they are stained they are mounted with DPX mountant film side down on glass slides

10. Automated Method The manufacturer’s instructions should be followed unless local experience has demonstrated that variation of the recommended technique achieves better results.

11. STAINING OF BLOOD SMEAR Blood smears are routinely stained by one of the Romanowsky stains: May- Grunwald - Giemsa stain Jenner stain Wright's stain Leishman's stain Field's stain

12. Two main components of all Romanowsky stains are an basic dye and a acidic dye • Basic or cationic dye : It is positively charged and binds to anionic sites and imparts blue-grey color to nucleic acids, nucleoproteins, and granules of basophils. Examples: methylene blue, azure B. • Acidic or anionic dye : It is negatively charged and binds to cationic sites and imparts orange-red color to hemoglobin and eosinophil granules. Example: eosin Y.

13. A well-stained smear is pink in color in thinner portion and purple-blue in thicker portion. The RBCs are spread uniformly without overlap and appear pink red in colour. Excess blue coloration can be due to : ( i ) excessively thick smear, (ii)low concentration of eosin, (iii) impure dyes, (iv) too long staining time, (v) inadequate washing, (vi) Excessive alkaline pH of stain, buffer, or water

14. Excess red coloration can be due to : impure dyes or incorrect proportion of dyes, excessive acid pH of stain, buffer, or water (as the red cells take up more acid dye i.e. eosin), (iii) too short staining time, (iv) excessive washing.

15. STAINING METHODS

16. 1.Leishman stain 2 MINS 5-7 MINS 2 MINS 2 mins 5 - 7 mins

17. 2. FIELD’S STAIN 10-15 SEC 1 MIN 5 SEC

18. 3. May-Grunwald-Giemsa stain

19. 5-10 MINS 15 MINS 10-15 MINS 2-5 MINS

20. 4. Jenner– Giemsa stain 4 MINS 7-10 MINS

21. A well-stained smear shows following features: • Red cells : pink-red or deep pink • Polychromatic cells (Reticulocytes): Gray-blue • Neutrophils : Pale pink cytoplasm; mauve-purple granules • Eosinophils : Pale-pink cytoplasm; orange-red granules • Basophils : Blue cytoplasm; dark blue-violet granules • Monocytes : Gray-blue cytoplasm; fine reddish (azurophil) granules • Small lymphocytes : Dark blue cytoplasm • Platelets : Purple • Nuclei of all cells : Purple-violet

22. EXAMINATION OF PHERIPHERAL SMEAR

23. A peripheral blood smear has three parts: Head, body,and tail . A blood smear should be examined in an orderly manner.

24. Examination of peripheral smear 1. RBC Size Colour Shape Inclusions Abnormal cells 2. WBC Total count Differential count Abnomal WBC 3. Platelets Counts Abnormality 4. Parasites

25. Red Blood Cells Best examined in an area where they are just touching one another. Size : 7-8 μ m round with smooth contours, stain deep pink at the periphery and paler in the center. Area of central pallor is about 1/3rd the diameter of the red cell. Size of a normal red cell corresponds roughly with the size of the nucleus of a small lymphocyte.

26. RBCs Abnormalities ANISOCYTOSIS Different SIZES of RBCs POIKILOCYTOSIS Different SHAPES of RBCs

27. VARIATION IN SIZE Microcyte Smaller than a normal cell; mean corpuscular volume usually <80 fL ; has a central pallor; normochromic or Seen inhypochromic Iron deficiency anemia Thalassemias Lead poisoning Anemia of chronic disease Sideroblastic anemia

28. Macrocyte Large cell, mean corpuscular volume usually >100 fL; usually normochromic; may be round or oval; cytoplasm is pink-red Seen in Liver disease (round macrocytes seen) Megaloblastic anemias (oval macrocytes seen) Myelodysplastic syndrome Acute blood loss Chemotherapy

29. VARIATION IN COLOUR H ypochromia Decrease in hemoglobin content of RBCs increase in central pallor>1/3rd decrease in MCV and MCHC seen in iron deficency anemia thalassemia

30. H yperchromia Red cells stained deeply have less or absent central pallor increase in MCH seen in megaloblastic anemia her e ditary spherocytosis

31. A nisochromia Presence of hypochromic and normochromic cells in same film also called as dimorphic anemia seen in sideroblastic anemia some weeks after iron therapy for iron def anemia hypochromic anemia after transfusion with normal cells

32. P olychromasia When RBC are delivered to the peripheral circulation prematurely appears diffusely basophilic and are grey blue in color and usually larger than normal red cell. The basophilic color is due to the RNA residue involved in hemoglobin synthesis. Polychromatic cells are actually reticulocytes. Any clinical condition in which marrow is stimulated particularly RBC regeneration will produce a polychromatic blood picture. The degree of polychromasia is a excellent indicator of therapeutic effectiveness when patient is given iron or vitamin therapy as treatment of anemia

33. Nucleated red blood cells (NRBCs) NRBCs are premature erythrocyte precursors that reside in bone barrow of humans of all ages as an element of erythropoiesis. They are rarely present in healthy adults circulatory system but can be found circulating in fetuses and neonates. Causes of NRBCs to be release into blood rapid blood loss damage or stress to the bone marrow If there are >10NRBCs/ 100 WBC then corrected TLC is given by the formula Corrected TLC = TLC ×100_________ NRBCs per 100 WBCs + 100

34. VARIATION IN SHAPE Variation in shape is called Poikilocytosis It is of following types - Sickle cell Spherocyte Target cell Echinocyte Bite cell Tear drop cell Elliptocyte Acanthocyte S t omatocyte Schistocyte

35. S ickle cell (Drepanocyte) Elongated or boat shaped with pointed ends cell due to polymers of abnormal hemoglobin; causing the cell to take on an irregular shape; usually lacks a central pallor Seen in Hemoglobinopathies

36. Sickling test procedure 1. Place one drop of the blood to be tested in a glass slide. 2. Add 1-2 drops of sodium metabisulfite to the drop of blood and mix well with an applicator stick. 3. Place a cover glass on top of the sample and press down lightly on it to remove any air bubbles and to form a thin layer of the mixture. Wipe of the excess sample. 4. Carefully rim the cover gl a ss with the petroleum jelly, completely sealing the mixture under the cover slip. 5. Examine the preparation for the present of sickle cells after one hour using 40 X objective . In some instances, the red blood cells may take on a holly -leaf form. This shape is found in sickle cell trait and when present ,the test is reported as positive. 6. If there is no sickling present at the end of one hour , allow the preparation to stand at room temperature for 24hrs , and examined at that time.

37. S pherocyte nearly spherical , diameter is smaller than normal no central pallor Seen in her e d i tary spher o cytosis some cases of autoimmune haemolytic anemia direct physical or chemical injury hypersplenism

38. T arget cell ( Codocyte )

39. Keratocyte (Horn cell) Cell with projections (usually two) that resemble horns Seen in Microangiopathic hemolytic anemia Glomerulonephritis Waring Blender syndrome Pyruvate kinase deficiency

40. E chinocytes (Burr cell) Also called crenated cells Numerous, short, regular projection Commonly occur as an artifact during preparation of film Seen in Renal insufficiency Pyruvate kinase deficiency Stored blood Severe dehydration Burns

41. A cant h ocytes Red cells with small no of spicules of inconstant length, thickness and shape, irregularly disposed over the surface. Seen in Abnormal phospholipid metabolism Abetalipoproteinemia Inherited abnormalities of red cell membrane protein Splenectomy

42. Bite cell (Degmacyte) It is a part of red cell is bitten off by the splenic macrophages Seen in G6PD Unstable hemoglobinopathies Drug induced anemias

43. Tear drop c ell (Dacrocyte) Pear-shaped cell rounded at one end and tapered at the other Seen in Extramedullary hematopoiesis (myelofibrosis, myelophthisic anemia) Megaloblastic anemia Thalassemia Hypersplenism

44. Elliptocyte Oval-shaped cell (may be slightly egg, rod, or pencil shaped); hemoglobin is concentrated at two ends; normal central pallor Seen in Hereditary elliptocytosis Iron deficiency anemia Megaloblastic anemia Thalassemia Sideroblastic anemia

45. S t omatocyte Red cells with central biconcave area appears slit like in dried film. Wet film it appears as cup- shaped. Seen in Hereditary stomatocytosis Alcoholism Obstructive liver disease Cirrhosis

46. S chistocyte these are fragmented erythrocytes smaller than normal red cells and varying shapes seen in Thalessemia Microangiopathic hemolytic anemias Traumatic hemolytic anemia Direct thermal injury

47. RED CELL INCLUSIONS Basophilic stippling (Punctate basophilia) Howell - jolly Bodies Heinz body Cabot Rings Protozoan inclusions Rouleaux formation

48. Basophilic Stippling Coarse, deep blue inclusions; irregularly aggregated or clumped ribosomes throughout the cell; mitochondria and siderosomes may also aggregate Seen in Altered hemoglobin biosynthesis Lead intoxication Thalassemia Megaloblastic anemia Alcoholism Sideroblastic anemia Pyrimidine-5′-nucleotidase deficiency

49. Howell Jolly Bodies Small ,round , purple -staining nuclear remnants Seen in Megaloblastic anemia Hemolytic anemia Postsplenectomy Sickle cell anemia

50. C abot ring Oval or figure eight–shaped inclusion; red-violet; usually one per cell; consists of nuclear remnants or part of the mitotic spindle Indicate impaired erythropoiesis Seen in Megaloblastic anemia Lead poisoning

51. P appenhiemer bodies Small, irregular, pale blue– to dark-staining granules; usually found on the periphery of the cell and in groups; smaller than Howell-Jolly bodies; represent siderosomes , which stain positive with Perls’ Prussian blue stain and indicate iron content Seen in Disturbed hemoglobin synthesis Sideroblastic anemia Dyserythropoietic anemias Thalassemia Myelodysplastic syndrome

52. H einz bodies Seen on supravital stains ( brilliant green , crystal violet or new methylene blue ) Not seen on Romanowsky stain. Purple, blue, large, single or multiple inclusions attached to the inner surface of the red blood cell. Consists of denatured globin produced by the destruction of hemoglobin Seen in Drug-induced anemias Thalassemia Glucose-6-phosphate dehydrogenase deficiency Unstable hemoglobinopathies

53. R oule au x formation Short or long stacks of cells (three or four or more) resembling coins; often a blue-staining background is also present Seen in Hyperproteinemia Multiple myeloma Macroglobulinemia Increased fibrinogen (infection, pregnancy)

54. A gglutination Random masses or clusters of cell Seen in Exposure to a variety of antibodies Hemolytic anemia (autoimmune) Atypical pneumonia Staphylococcal infections Trypanosomiasis Cold agglutinin dise ase

55. RETICULOCYTES Reticulocytes are young or juvenile red cells released from the bone marrow into the bloodstream and that contain remnants of ribonucleic acid (RNA) and ribosomes but no nucleus. Reticulocyte count is performed to assess erythropoietic activity of the bone marrow

56. USES As one of the baseline studies in anemia with no obvious cause To diagnose anemia due to ineffective erythropoiesis To assess response to specific therapy in iron deficiency and megaloblastic anemias . 4. To assess response to erythropoietin therapy in anemia of chronic renal failure.

57. Staining of Reticulocytes 15 MINS

58. Reticulocyte counting Miller ocular disk is inserted in the eyepiece; it divides the field into two squares (one nine times larger in size than the other). Reticulocytes are counted in both the squares red cells are counted in the smaller square.

59. FORMULAS 1 .Reticulocyte Number of reticulocytes counted percentage = ———————————————— × 100 Number of red cells counted Total reticulocytes counted in square A 2.Reticulocyte count = —————————————————— ×100 Total red cells counted in square B × 9 3. Absolute reticulocyte count = Reticulocyte percentage × Red cell count Normal: 50,000 to 85,000/ cmm 4. Corrected reticulocyte count ( Reticulocyte index) PCV of patient = Reticulocyte percentage × ———————— Normal PCV

60. WHITE BLOOD CELLS

61. Granulocytes Agranulocyte it contains membrane bound granules which stains differently with stains apperantely absent granules but contains nonspecific azurophillic granules eg ; neutrophills basophills eosinophills eg ; lymphocytes monoytes macrophages

62. Segmented Neutrophil or Polymorphonuclear neutrophil Size: 10–16 μ Nucleus Shape: 2–5 lobes connected by a very narrow filament; nuclear indentation is greater than one-half its diameter N/C Ratio: 1:3–1:5 Color: Dark purple Chromatin: Heavily clumped Nucleoli: None Cytoplasm Color: Light pink to bluish Contents: Many small, evenly distributed pink to rose-violet granules

63. B and forms Neutrophils has either a strand of nuclear material thicker than a filament connecting the lobes, or a U- shaped nucleus of uniform thickness. Up to 8% of circulating neutrophils are unsegmented or partly segmented ('band' forms )

64. Band cells 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

65. CYTOPLASMIC INCLUSIONS Toxic granules Heavy, coarse, dark-blue primary cytoplasmic granules Strong peroxidase reactivity Seen in Infections Burns Drug intoxication Inflammation Growth factor thera py

66. A lder R eilly anomaly Dense blue cytoplasmic granules consisting of stored mucopolysaccharides and sphingomyelin Normal nuclear maturation Seen in Mucopolysaccharidoses (e.g., Hurler’s syndrome, Hunter’s syndro me)

67. Chediak- Higashi syndrome Granules are giant scanty azurophillic functional defect occur also seen in other leukocytes like lymohocytes

68. D ohle bodies small round or oval pale blue grey structure found at the periphrey of neutrophill contains ribosomes and endoplasmic reticulum seen in bacterial infection,infla m mation and administration of G-CSF during pregnancy

69. NUCLEAR SEGMENTATION Hypersegmentation Presence of neutrophils with six or more lobes or the presence of more than 5% of neutrophils with five lobes. Seen in Chronic infections B12 deficiency Folic acid deficiency Myelodysplastic syndromes Hereditary hypersegmentation Long term infections

70. Pelger-Huët anomaly Benign inherited condition. Neutrophil nuclei fail to segment properly. Majority of circulating neutrophils have only two discrete equal-sized lobes connected by a thin chromatin bridge

71. Eosinophil Size : 15–16 μm Nucleus Shape: 2–3 lobes N/C Ratio: 1:3–1:5 Color: Dark purple Chromatin: Heavily clumped Nucleoli: None Cytoplasm Color: Pink-blue Contents: Many large, round, uniform reddish-orange granules

72. EOSINOPHILIA Absolute Eosinophilic count >600/microlitre Causes- 1. Allergic diseases : Bronchial asthma , rhinitis , urticaria, drugs. 2. Skin diseases: Eczema, pemphigus, dermatitis herpetiformis. 3. Parasitic infection with tissue invasion: Filariasis, trichinosis, echinococcosis. 4. Hematologic disorders: Chronic myeloproliferative disorders, Hodgkin's disease, peripheral T cell lymphoma. 5. Carcinoma with necrosis.

73. Basophil Basophils are seen rarely on normal smears. They are Size - 9-12 μm round to oval Nucleus Shape: 2 lobes usually obscured by granules N/C Ratio: 1:3–1:5 Color: Dark purple Chromatin: Heavily clumped Nucleoli: None Cytoplasm Color: Pink-blue Contents: Few dark blue–black granule

74. Increased numbers of basophils in blood (>100/ m l) Causes- chronic myeloid leukemia , 2) polycythemia vera, 3) Idiopathic myelofibrosis, 4) basophilic 5) Leukemia 6) myxedema , 7) hypersensitivity to food or drugs. BASOPHILIA

75. Monocyte Monocyte is the largest of the leukocytes (15-20 μm ). irregular in shape, with oval or clefted (kidney-shaped) nucleus and fine, delicate chromatin. Cytoplasm is abundant, blue grey with ground glass appearance Often contains fine azurophil granules and vacuoles.

76. Monocytosis This is an increase in the absolute monocyte count above 1000/μ l. Causes 1. Infections: Tuberculosis, subacute bacterial endocarditis, malaria, kala azar. 2. Recovery from neutropenia. 3. Autoimmune disorders. 4. Hematologic diseases: Myeloproliferative disorders, monocytic leukemia , Hodgkin's disease. 5. Others: Chronic ulcerative colitis, Crohn's disease, sarcoidosis.

77. Lymphocyte Small lymphocyte : Size : 7-8 μ m thin rim of deep blue cytoplasm. The nucleus is round or slightly clefted with coarsely clumped chromatin Large lymphocyte : Size :10-15 μm more abundant, pale blue cytoplasm, which may contain a few azurophil granules. Nucleus is oval or round and often placed on one side of the cell.

78. Reactive lymphocytes Have slightly larger nuclei with more open chromatin Abundant cytoplasm that may be irregular. Seen in -infectious mononucleosis , viral infections

79. Türk' cell Türk' cell ( immunoblasts )- Transformed lymphocyte seen in bacterial and viral infection Size 10-15 µm Nucleus- Round, Large nucleolus, and abundant, deeply basophilic cytoplasm

80. PLATELET MORPHOLOGY SIZE : 1–3 μ m in diameter Irregular in outline with fine red granules that may be scattered or centralised .

81. Thrombocytopenia Decrease production Aplastic anemia Acute leukemia Viral infections *Parvovirus *CMV Increased destruction Immune thrombocytopenia Idiopathic thrombocytopenic purpura (ITP) Neonatal alloimmune thrombocytopenia (NAITP) Disseminated intravascular coagulation (DIC) Hypersplenism Pseudothrombocytopenia - due to clumpping of pl a tel ets in EDTA bulb Thromocytosis Reactive thrombocytosis Post infection Inflammation Juvenile rheumatoid arthritis Collagen vascular disease Essential thrombocythem ia

82. GIANT PLATELET PLATELET SATELLITISM Adhesion of platelet to neutrophil

83. EXAMINATION OF BLOOD FILMS FOR PARASITES PARASITES DETECTABLE IN BLOOD : Plasmodia Leishmaniae , Babesiae , Trypanosomes Microfilariae

84. STAINING FOR PARASITES Field staining: 1. Dip the slide with the dried film on it into Stain A for 3 s. 2. Dip into a jar of tap water for 3s with gentle agitation. 3. Dip into Stain B for 3 s. 4. Wash gently in tap water for a few seconds until all excess stain is removed. 5. Drain the slide vertically and leave to dry. Do not blot. Leishman stain 1. Make a thin film and air-dry rapidly. 2. Place the film on a staining rack, flood with Leishman stain and leave for 30 s to 1 min to fix. 3. Add twice as much buffered distilled water (preferably from a plastic wash bottle because this permits better mixing of the solution), pH 7.2. 4. Leave to stain for 10 min. 5. Wash off stain with tap water.

85. Ring stage of  P. Falciparum . P. falciparum  gametocyte Maurer dots of P. Falciparum

86. Ring form with chromain dot of P. vivax Macrogametocyte of P.vivax

87. Leishmaniasis ( Leishman - Donovan bodies)

88. A frican trypanosomiasis (T bruzi) American trypanosomiasis (T cruzi)

89. Microfilaria

90. REFER E NCES -Essentials of clinical pathology - Shirish M Kawthalkar -Practical Haematology -Dacie and Lewis -Atlas and text of haematology - Dr. Tejinder Singh -Anderson’s atlas of haematology