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1. Complete Blood Count Lab-1

2. Introduction The complete blood count (CBC) is one of the most commonly ordered blood tests. The complete blood count is the calculation of the cellular (formed elements) of blood. These calculations are generally determined by special machines that analyze the different components of blood in less than a minute. A major portion of the complete blood count is the measure of the concentration of white blood cells , red blood cells , and platelets in the blood.

3. When CBC is ordered? CBC may be ordered when a person has any number of signs and/or symptoms that may be related to disorders that affect blood cells. When an individual has fatigue or weakness  Infection.  Inflammation. Bruising, or bleeding. To help diagnose the cause and/or determine its severity. Monitor disease and treatment conditions.

4. Parameters of CBC The complete blood count, or CBC, lists a number of many important values. Typically, it includes the following: White blood cell count (WBC or leukocyte count) WBC differential count Red blood cell count (RBC or erythrocyte count) Hematocrit ( Hct ) Hemoglobin ( Hb ) Mean corpuscular volume (MCV) Mean corpuscular hemoglobin (MCH) Mean corpuscular hemoglobin concentration (MCHC) Red cell distribution width (RDW) Platelet count Mean Platelet Volume (MPV)

5. White Blood Cells Count The normal number of WBCs in the blood is: 4 - 11 X 10 3 /µL , or 4 - 11 X 10 9 / L Causes of Leukocytosis Causes of Leukopenia Infection, most commonly bacterial  Bone marrow disorders or damage Inflammation Autoimmune conditions Leukemia, Myeloprolifrtive disorders Severe infections (sepsis) Allergies, asthma cancer that spread to the bone marrow Tissue death (trauma, burns, heart attack) Diseases of immune system (e.g.,HIV) Intense exercise or severe stress

6. White Blood Cell Differential WBC Normal range Causes of Increasing Causes of Decreasing Neutrophils 40-80% of WBCs Neutrophilia mainly associated with bacterial infections. Neutropenia may occur due to viral infections and some medications. Lymphocytes 20-40% of WBCs Lymphocytosis mainly associated with acute viral infections, Lymphocytic leukemia, Connective tissue diseases Lymphopenia may due to bacterial, and fungal infections. Monocytes 2-10% of WBCs Monocytosis : often occur due to chronic inflammation, during the recovery period after severe infections, infectious mononucleosis. Monocytopenia may develop due to acute infections, stress, bone marrow failure. Eosinophils 1-6% of WBCs Eosinophilia mainly associated with allergic diseases, and parasitic infections. Eosinopenia found during the acute stages of all acute infectious diseases. Basophils 0-1% of WBCs Basophilia caused mainly by allergic reaction Basopenia has no diagnostic value.

7. Absolute Differential Count Absolute count = Relative(%)x WBCs cout ÷ 100 e.g : WBCs= 4000 c/microliter, Neutrophil= 45% Thus: the absolute count of neutrophil = 45x 4000 ÷ 100 = 1800 c/ microliter.

8. Absolute Differential Count TEST RESULT UNITS REF RANGE WBC 5.2 x 1000/mm 3 4 - 11 RBC 3.81 L x 10 6 /mm 3 4.5 - 5.5 HGB 14.5 g/dL 13 - 17 HCT 41.2 % 40- 52 MCV 98 H fl 80 - 97 MCH 33.7 H pg 27.5 - 33.5 MCHC 35.3 % 32.0 - 36.0 RDW 11.8 % 11.0 - 15.0 PLT 172 x 1000/mm 3 140 - 390 MPV 7.6 fl 7.5 - 11.5 NEUT % 40.1 % 40 - 80 LYMPH % 46.1 % 20 - 40 MONO % 12.9 % 2- 10 EOS % 0.6 % 1- 6 BASO % 0.3 % 0.0 - 2.0 NEUT, ABS 2085 cells/mm 3 1650 - 8000 LYMPH, ABS 2397 cells/mm 3 1000 - 3500 MONO, ABS 671 cells/mm 3 40 - 900 EOS, ABS 31 cells/mm 3 30 - 600 BASO, ABS 16 cells/mm 3 0 - 125 5.2 x 1000 = 5200 5200 x .401 = 2085

9. Platelet Count (PLT, Thrombocyte Count)   The normal range = 150 - 400 X 10 3 /µL or 150 - 400 X 10 9 / L Thrombocytosis caused by: Cancer (lung, gastrointestinal, breast, ovarian, lymphoma) Rheumatoid arthritis, inflammatory bowel disease, lupus Iron deficiency anemia Hemolytic anemia Myeloproliferative disorder (e.g., essential thrombocythemia ) Thrombocytopenia caused by Viral infection (mononucleosis, measles, hepatitis) Rocky mountain spotted fever, Sepsis Platelet autoantibody Drugs (acetaminophen, quinidine, sulfa drugs) Leukemia, lymphoma, Myelodysplasia Chemo or radiation therapy

10. Erythrocyte count (RBCs) Normal RBC range is: Males: 4.5-5.5 X 10 6 c/ µL or 4.5-5.5 X 10 12 c/ L Females: 4.0-5.0 X 10 6 c/ µL or 4.0-5.0 X 10 12 c/ L RBCs, is generally, decreased in all types of anemia, and increased in all causes of polycythemia.

11. Hemoglobin  Hemoglobin measures the amount of oxygen-carrying protein in the blood. Normal values:  In adult males 13-17 g/ dL = 130-170 g/L. In adult, non-pregnant females 11.5-15 g/ dL =115-150 g/L Hb , is generally, decreased in all types of anemia, and increased in all causes of polycythemia.

12. Hematocrit (Hct), Packed Cell Volume, (PCV) Hematocrit is the ratio of the volume of red blood cells to the volume of blood plasma. Normal values ( Hct ):  0.40-0.52 L/L= 40-52% in adult males 0.37-0.47 L/L= 37-47% in adult females. Hct , is generally, decreased in all types of anemia, and increased in all causes of polycythemia.

13. Red Blood Cell Indices Red blood cell indices are calculations that provide information on the physical characteristics of the RBCs: Mean corpuscular volume (MCV) is a measurement of the average size of RBCs. Mean corpuscular hemoglobin (MCH) is a calculation of the average weight (content) of hemoglobin in red blood cells. Mean corpuscular hemoglobin concentration (MCHC) is a calculation of the average percentage of hemoglobin in each individual RBC. Red cell distribution width (RDW), is a calculation of the variation in the size of RBCs.

14. Mean Corpuscular Volume (MCV) MCV = Hct (%) ÷ RBCs count (cell/L) x 10 Expressed in fl (femtoliter). 1 liter = 10 15 fl , thus 1 fl= 10 -15 Liter e.g.: if the Hct 45%, and the RBCs is 5 X 10 12 per liter. MCV = 45 ÷ 5 X 10 = 90 fl (femtolitre) Normal value for the MCV : 80- 95 fl If the MCV is less than 80 fL, means (Microcytic) If the MCV is greater than 95 fl, means (Macrocytic) If the MCV is within the normal range, the RBCs are Normocytic

15. Mean Corpuscular Hemoglobin (MCH) MCH = Hb conc (g/L) ÷ RBCs count (c/L) Expressed in picogram ( pg ) 1 gram = 10 12 pg , thus 1 pg = 10 -12 gram e.g : if there are 150g of Hb and 5 X 10 12 red cells per litre , MCH = 150 ÷ 5 = 30pg ( picograms ) Normal value for the MCH: 27-32 pg An MCH lower than 27 pg (Hypochromic) An elevated MCH ( hyperchromic ) occurs in some cases of spherocytosis .

16. Mean Corpuscular Hemoglobin Concentration (MCHC) MCHC = e.g : if the Hb concentration is 150g/L and the Hct is 0.45 MCHC = 150 ÷ 0.45 = 333g/L =33.3% (g/dl) Normal value for the MCHC : 32-35.5 % or 320-355 g/L Hemoglobin in g/L Hematocrit L/L

17. Red cell distribution width (RDW) RDW is an index of the variation of red cell size (volume) in a specimen of blood Normal range: 13 ± 1.5 % Low value indicates uniformity in size of RBCs High value indicates a mixed population of small and large RBCs; ( anisocytosis ).

18. Advantages of having RDW : 1. Recognize RBC abnormality from CBC 2. Assist in differential diagnosis 3. Following the course of a disease

19. Automated Haematology Analysers - Lab -2

20. Current hematology analyzers use a combination of light scattering , electrical impedance , fluorescence , light absorption , and electrical conductivity methods to produce complete red blood cell, platelet, and leukocyte analyses. Introduction:

21. Electrical Impedance

22. Using this technology, cells are sized and counted by detecting and measuring changes in electrical resistance when a particle passes through a small aperture. This is called the electrical impedance principle of counting cells. A blood sample is diluted in saline , a good conductor of electrical current, and the cells are pulled through an aperture by creating a vacuum. Two electrodes establish an electrical current. The Coulter Principle

23. Low-frequency electrical current is applied to the external electrode and the internal electrode. Electrical resistance or impedance occurs as the cells pass through the aperture causing a change in voltage. This change in voltage generates a pulse . The number of pulses is proportional to the number of cells counted. The size of the voltage pulse is also directly proportional to the volume or size of the cell .

24. Whole blood is aspirated, diluted, and then divided into two samples . One sample is used to analyze the red blood cells and platelets while the second sample is used to analyze the white blood cells and hemoglobin . Electrical impedance is used to count the white blood cells, red blood cells, and platelets as they pass through an aperture. The amplitude of each pulse is directly proportional to the cell volume. Performance

25. In the RBC chambe r, both the RBCs and the platelets are counted and discriminated by electrical impedance Particles between 2 and 20 fL are counted as platelets, and those greater than 36 fL are counted as RBCs. Lyse reagent is added to the diluted sample and used to count the white blood cells. The lysing reagent also causes WBC's membrane collapse around the nucleus, so the counter actually measures the nuclear size . After the white blood cells have been counted and sized, the remainder of the lysed dilution is transferred to the Hgb Flow Cell to measure Hemoglobin concentration.

26. Specimen Requirements: Whole blood collected in an EDTA tube. Samples are stable at room temperature for eight hours. Analysis Modes: 1- Whole blood mode 2- Pre-diluted mode

27. Sources of Errors in cell count 1- Cold agglutinins Cause low red cell counts and high MCVs, hematocrits and MCHCs are also incorrect. This can be prevented by warming the sample in a 37°C

28. 2-Fragmented or very microcytic red cells These may cause red cell counts to be decreased. Sources of Errors in cell count

29. 3-Platelet clumps and platelet satellitosis : These cause falsely decreased platelet counts. Sources of Errors in cell count

30. 4-Giant platelets: These are platelets that approach or exceed the size of the red cells, which cause a decrease in plts count. Sources of Errors in cell count

31. 4-Nucleated red blood cells: These interfere with the WBC on some instruments by being counted as white cells/lymphocytes. Sources of Errors in cell count

32. Anything that will cause turbidity and interfere with a Spectrophotometry method. Examples: a very high WBC or platelet count, lipemia, and hemoglobin's that are resistant to lysis, such as hemoglobin's S and C. Sources of Errors in Measuring Hemoglobin

33. Anything affecting the red cell count or volume measurement will affect the hematocrit. Sources of Errors in Hematocrit

34. Correlating Hemoglobin and Hematocrit Values The hemoglobin times three roughly equals the hematocrit in most patients. Example : 14.8 x 3 = 44 (patient's hematocrit result is 45%) 11.0 x 3 = 33 (patient's hematocrit result is 32 %) The exception to this rule is in patients with hypochromic red cells. These patients will have hematocrits that are more than three times the hemoglobin.

35. In most automated systems, the complete blood count is numerically reported.. The differential is numerically recorded and then graphically displayed How Data are Reported?

36. RBC and Platelet Histograms The black line represents normal cell distribution. The red line on the RBC histogram graphically represents a Microcytic red cell population.

37. Red Cells Histogram normal red cell histogram displays cells form (36- 360 ) fl (24- 36 fl ) flag may be due 1- RBCs fragments 2- WBC's fragments 3- Giant plts 4- Microcyte Shift to right : - Leukemia - Macrocytic anemia - Megaloblastic anemia Shift to left : - Microcytic anemia Bimodal - Cold agglutinin - IDA, Megaloblastic anemia with transfusion. -Sideroblastic anemia. Trimodal - Anemia with transfusion

38. Plts Histogram Normal platelet histogram displays cells from (2-20 fl). (0-2) Air Babbles Dust Electronic and Electrical noise Over 20 fL Microcyte Scishtocyte WBC's fragments Giant Plts Clumped plts

39. The histogram is a representation of the sizing of the leukocytes. The differentiation is as follows: Leukocyte Histogram Analysis

40. WBC Unusual RBC abnormalities that resist lysis Nucleated RBCs Fragmented WBCs Unlysed particles greater than 35 fL Very large or aggregated plts Specimens containing fibrin, cell fragments or other debris (esp pediatric/oncology specimens Interferences that may cause Erroneous results RBC Very high WBC (greater than 99.9) High concentration of very large platelets Agglutinated RBCs. RBCs smaller than 36 fL. Specimens containing fibrin, cell fragments or other debris (esp pediatric/oncology specimens

41. Interferences that may cause Erroneous results Hgb Very high WBC count. Severe lipemia . Heparin. Certain unusual RBC abnormalities that resist lysing. Anything that increases the turbidity of the sample such as elevated levels of triglycerides and High bilirubin. MCV Very high WBC count. High concentration of very large platelets. Agglutinated RBCs RBC fragments that fall below the 36 fL threshold Rigid RBCs.

42. Interferences that may cause Erroneous results RDW Very high WBC High concentration of very large or clumped platelets RBCs below the 36 fL threshold Two distinct populations of RBCs RBC agglutinates Rigid RBCs Plt Very small red cells near the upper threshold Cell fragments Clumped platelets Cellular debris near the lower platelet threshold

43. Interferences that may cause Erroneous results MPV Known factors that interfere with the platelet count and shape of the histogram Known effects of EDTA Hct Known factors that interfere with the parameters used for computation, RBC and MCV MCH Known factors that interfere with the parameters used for computation, Hgb and RBC MCHC Known factors that interfere with the parameters used for computation, Hgb, RBC and MCV

44. Plts < 40,000 Check the integrity of the specimen (look for clots, short draw, etc.) Confirm count with smear review for clumps, RBC fragments, giant platelets, very small RBCs WBC ++++ Dilute 1:2 with Isoton or further until count is within linearity (for final result, multiply diluted result by dilution factor); subtract final WBC from RBC; perform spun hct , calculate MCV from correct RBC & Hct (MCV = Hct /RBC x 10), do not report HGB, MCH, MCHC. Plt counts are not affected by high WBC. Add comment, “Unable to report Hgb , MCH, MCHC due to high WBC.” Handling abnormal results

45. Plt ++++ Check smear for RBC fragments or microcytes. If present, perform manual plt count. If not present, dilute specimen 1:2 with Isoton or further until the count is within linearity , multiply the diluted result by the dilution factor. RBC > 7.0 Dilute 1:2 with Isoton or further until the count is within linearity, multiply dilution result by dilution factor; perform spun hct, review Hgb, recalculate MCH, MCHC Handling abnormal results

46. Approach to anaemia Lab-3

47. Mature red blood cells have non nucleus and their colour cytoplasm is pink, central pallor about 1/3 of the cell. They have not mitochondria The red blood cell under the microscope Image shows Mature red blood cells Normal RBCs are biconcave, smooth, elastic and allow for simplicity in movement (i.e. from side to side), in small blood vessels. The life span of the normal RBC is about 120 days .

48. Blood smear demonstrating features of a patient with iron deficiency anaemia, including hypochromic, microcytic RBCs Blood smear demonstrating features of an individual with thalassemia demonstrates hypochromic, microcytic RBCs , variously-shaped (poikilocytosis) RBCs. * Poikilocytosis = variation in shape of RBCs Clinical conditions for patients with hypochromic and microcytic picture:- Iron Deficiency Anaemia of chronic disease Sideroblastic anaemia Lead Poisoning Thalassaemia MCV

49. Iron profile Disease Serum iron TIBC % Transferin saturation Ferritin Iron Deficiency Low High Low Low Thalassaemia Normal or increased Normal Normal or increased Increased Anaemia of chronic disease Low Low Low Normal/High Sideroblastic anaemia Normal/High Normal/Low High High Lead Poisoning High Normal High Normal A low iron with a high transferrin or total iron binding capacity (TIBC) is usually due to iron deficiency. In chronic diseases, both iron and transferrin or TIBC are typically low. High levels of serum iron can occur as the result of multiple blood transfusions, iron injections into muscle, Lead Poisoning, liver disease or kidney disease.

50. Blood cells morphology Morphology Clinical conditions Megaloblastic anaemia Liver disease 3. Alcoholism. - Nucleus : Round or irregular; - Nucleus/ Cytoplasm : 5:1; - Chromatin : Fine and closely meshed. This chromatophilic area is caused by premature Hb ; -Nucleoli: multiple (not visible); -Cytoplasm: deep blue colour, nongranular with perinuclear hallo Vitamin B12 deficiency Folic acid deficiency Congenital diserythropnaemia Picture shows basophilic Promegaloblast Picture shows Image shows Macrocytes MCV

51. Picture shows basophilic Promegaloblast . ( -Nucleus: Round or irregular -Nucleus/ Cytoplasm: 5:1 -Chromatin: Fine and closely meshed. This chromatophilic area is caused by premature Hb . -Nucleoli: multiple (not visible) -Cytoplasm: deep blue colour, nongranular with perinuclear hallo Picture shows Polychromatophilic megaloblast -Nucleus: Round and central Nucleus/ Cytoplasm: 2:1 Chromatin: Minimal clumbing , loosely defined. -Nucleoli: Not visible. -Cytoplasm: Blue-grey to pink-grey. More cytoplasm than in normoblastic cell. Clinical conditions for both basophilic Promegaloblast and Polychromatophilic megaloblast Vitamin B12 deficiency Folic acid deficiency Congenital diserythropnaemia

52. RBCs have a hard time making DNA, because there is a lack of B12 and/or folate , but RNA production proceeds normally. Thus, RBCs have normally maturing cytoplasm, but slowly-maturing nuclei. This means that the RBCs grow large before the nucleus becomes mature enough to signal division and therefore the RBCs end up being larger than normal. Nucleus looks more immature than the cytoplasm (hence the term “nuclear- cytoplasmic asynchrony) Hypersegmented Neutrophil :- Increased size and lobulation , six or more lobes. This condition is often indicative of reduced DNA synthesis Clinical conditions : Vitamins B12 deficiency or/and folate deficiency Anti-metabolite therapy or alcoholism Figures show megaloblastic anaemia . Macrocytes with Neutrophil hypersegmented .

53. Blood cells morphology Morphology Clinical conditions Picture shows Elliptocytes Hereditary Elliptocyosis Iron deficiency anaemia (In severe cases) Figure shows Heinz bodies ( HzB ) Description: Heinz body aggregates of oxidized denatured precipitated Hb within RBC. * Heinz body can be easily observed with supra-vital stains, such as new methylene blue . Glucose-6-Phosphate Dehydrogenase. Drug-induced anaemia 3. Chronic liver disease . 4. Hyposplenism or a splenia Description: Elliptocyte i s Oval-shaped (may be slightly egg, rod, or pencil shaped), Hb is concentrated at two ends; normal central pallor .

54. Blood cells morphology Morphology Clinical conditions 1. Sideroblastic anaemia 2. Lead poisoning 3. Arsenic poisoning 4. Beta- thalassaemia and alpha- thalassaemia 5. Alcoholism 6. Megaloblastic anaemia * Description: It is a small fragment of non-functional nucleus 1. Severe iron deficiency anaemia 2. A splenia 3. Hyposplenism 4. Pernicious anaemia 5. Underdeveloped spleen 6. Alcoholism 7. Splenectomized Images shows Howell-Jolly's body Basophilic Stippling is also called Punctuate Basophilia Cell type: Mature RBCs. Description : Coarse, deep blue inclusions, irregularly aggregated or clumped ribosomes throughout the cell, mitochnodria and siderosomes may also aggregate . Images show Basophilic Stippling

55. Morphology Clinical conditions Schistocyte A microangiopathic haemolytic anaemia (MAHA) Traumatic haemolytic anaemia . Waring Blender syndrome ( the shearing of erythrocytes by obstructions of the vascular bed, such as heartworms and disseminated intravascular coagulation Hereditary stomatocytosis Alcoholism Cirrhosis Rh -null disease (D -, C- , E-, c-, e-) Obstructive liver disease Stomatocyte Description Stomatocyte is RBC with an oval or rectangular area of central pallor, sometimes referred to as a "mouth". These cells have lost the indentation on one side Description: Schistocyte is irregular shape or fragment of cell; results from damaged membrane. Some of the irregular shapes appear as "helmet" cells. Such fragmented RBC's are known as " schistocytes "

56. Blood cells morphology Morphology Clinical conditions Images shows Acanthocyte Inherited lipid disorder (Individuals with abetalipoproteinaemia ) Neonatal hepatitis Pyruvate kinase deficiency Alcoholic cirrhosis  Individuals with spur cell haemolytic anaemia Picture shows Echinocyte (Burr cell) Stored blood Severe dehydration Pyruvate kinase deficiency Burns Renal insufficiency   Description: Echinocytes  (also called burr cells) have serrated edges over the entire surface of the cell and often appear crenated in a blood smear.   Description: The cells appear contracted, dense, and irregular. Its formation depends on alteration of the lipid composition and fluidity of the RBC membrane. Acanthocyte is spherical and densely stained cell with 3 – 12 spicules of uneven length and width around the surface.

57. Description: The RBC is shaped like a teardrop. These cells have membrane abnormalities ( Poikilocytosis ) and both microcyte and macrocytes ( Anisocytosis ). Clinical conditions Myelofibrosis Megaloblastic anaemia Hypersplenism Metastatic carcinoma. They are seen when there is extramedullary erythropoiesis or with marrow disorders or marrow infiltration Image shows T eardrop cell  (dacrocyte) Figure shows Red cell autoagglutination Description: It is the process whereby RBCs clump together forming aggregates. This is due to the RBCs being coated on their surface by antibodies. Clinical conditions Autoimmune haemolytic anaemia Cold agglutinin disease Waldenström’s macroglobulinemia Trypansomiasis Infection with Mycoplasma Pneumonia or Infectious mononucleosis.

58. Description: RBC abnormalities in SC include target cells (double arrow), distorted RBCs due to the presence of SC crystals (single arrow), and sickle-like forms (double blunt arrow). Clinical conditions 1. Haemoglobin SC disease Picture shows Haemoglobin SC crystals Description: It is also called Codocyte . They  RBCs that have the appearance of a shooting target with a bullseye. In optical microscopy these cells appear to have a dark center (a central, hemoglobinized area) surrounded by a white ring (an area of relative pallor), followed by dark outer (peripheral) second ring containing a band of Hb . Clinical conditions Haemoglobinopathies ( thalassaemia , sickle cell anaemia ) Obstructive jaundice Iron deficiency anaemia Target cells ( Codocytes ,)

59. Picture shows Pinch cells Description: it is also called Knizocyte . Pinch cells have two concavities instead of the one seen with normal RBCs. Like normal erythrocytes, knizocytes show a clear central area, but this is crossed by a thin strip of Hb Clinical conditions of Pinch cells :- Haemolytic anaemia Haemoglobinopathies Pancreatitis Picture shows Blister cells Description: it is also called Pyknocyte , acell with a clearing on one side and a concentrated area of Hb on the other side Clinical conditions of Blister cells:- Infantile pyknocytosis Infantile viremia . Description: The hypochromic cell contains small blue granular, irregular-shaped inclusions on the periphery of the RBC. These iron-containing (ferritin) granules are called Pappenheimer bodies. The presence of non- heme iron in the granules is confirmed by A Prussian Blue stain. Clinical conditions Thalassaemia Sideroblastic anaemia Dyserythropoietic anaemias Myelodysplastic syndrome Pappenheimer body

60. Bite cells Description: Bite cell is called Degmacytes . It is RBC with peripheral single or multiple arcuate defects. Usually associated with spherocytes and blister cells. Semicircular area of cell removed by spleen Clinical conditions Normal individuals receiving large quantities of aromatic drugs containing amino, nitro, or hydroxy groups. Enzymopathies , most notably G6PD deficiency. Drug induced anaemias Thalassaemia and unstable haemoglobinopathies . Rouleaux Formation. Description: Stacking of RBCs occurs in the presence of increased amounts of fibrinogen, immunoglobulin or acute phase reactants in the serum. In contrast, normal red cell membranes are negatively charged and red cells will normally "repel" one another. In the presence of positively charged proteins, the red cells may be brought together in these stacks, known as rouleaux .  Clinical conditions Hyperproteinaemia . Multiple myeloma Macroglobulinaemia Increased fibrinogen (infection, pregnancy

61. The reticulocyte is the final stage of the development of RBC before full maturation. The presence of reticulocyte in normal blood in very low number (perhaps one reticulocyte per 100 mature RBCs). High number of the reticulocytes may be the product of pathological process or could be the body’s response to pregnancy, iron, B12 or folic acid therapy, or to blood loss. Reticulocytes are not always part of the FBC and therefore, if required, need to be specially requested. The final step of erythropoiesis is the maturation of reticulocyte into the mature erythrocyte, which then passes into the blood circulation. However, some reticulocytes pass directly into the circulation for an additional 1 to 2 days, following which they differentiate into mature RBCs.

62. The reticulocyte can be differentiated from the mature erythrocyte not only by its slightly large size, but also because it contains remnants of messenger RNA for Hb , detectable by supra vital special stain such as brilliant cresyl blue (see Figure 1) . However, although reticulocytes generally can not be specifically identified by conventional Romanowsky staining, a proportion do stain a slightly bluer colour than mature RBCs. This bluish tinge is referred to as polychromasia (see Figure 2 ).

63. Figure 1. Picture shows Reticulocytes, using vital stain. Description: They normally make up 1% of the total RBC count, but may exceed levels of 2.5% when compensating for anaemia (haemolytic anaemia). Figure 2. Picture shows Reticulocyte (Poly chromatophilic ), using Giemsa’s stain. Description: Non nucleus; Cytoplasm is pink with a tint of blue. It contains remnants of Golgi and Mitochondria and residual RNA Clinical conditions Low reticulocytes are found in: Chemotherapy Aplastic anaemia Pernicious anaemia BM malignancies Problems of erythropoietin production; various vitamin or mineral deficiencies High reticulocytes are found in ( Reticulocytosis ): Increased erythrocyte production (High erythropoiesis) Haemolytic anaemias

64. As this final step in RBCs maturation can take place in peripheral blood as well as in the BM, small number of reticulocytes (perhaps 0.5 - 1.5% of the entire red cell population) may be present in healthy blood. However, large numbers of reticulocytes, perhaps greater than 2.5%, imply an abnormality, such as in certain types of anaemia . Similarly nucleated red blood cells are seen so rarely in healthy adult blood that their presence is inevitably the result of a pathological process, although nucleated RBCs may rarely be seen in healthy neonatal blood (we will discuss it in detail later).

65. Erythrocyte sedimentation rate (ESR) This rate of "sinking" is known as the ESR and is an indirect measure of rouleaux formation. Conditions that increase the ESR include 1. Infection 2. Inflammation 3. Malignancy (since they are associated with increase in acute phase reactants) 4. Multiple myeloma (because of elevated immunoglobulin in the serum) 5. Anemia (decrease in the viscosity of blood). Conditions that decrease the ESR include 1. Polycythemia (because of the increased viscosity of the blood 2. The presence of abnormally shaped red blood cells (sickle cells e.g. sediment more slowly because of their abnormal shape)

66. Morphological Classification of anemia

67. A. Red cell membrane defects 1. Hereditary spherocytosis 2. Hereditary elliptocytosis B. Red cell enzyme defects ( Enzymopathies ) 1. Glucose-6-phosphate dehydrogenase deficiency 2. Pruvate kinase deficiency C. Haemoglobinpathies 1. Hb variants ( Hb S, C, E and D) 2. thalassaemia syndrome ( α and β thalasaemias) Classification of inherited haemolytic anaemia

68. Laboratory findings in extravascular hemolysis 1- Features of increase RBCs breakdown a- Serum bilirubin raised b- Urine urobilinogen increased. c- Fecal strecobilinogen increased. d- Serum haptoglobin absent. 2- Features of increased red cell production a- Reticulocytosis. b- Bone marrow erythroid hyperplasia. 3- Features of damaged red blood cells a- Red cell morphology ( spherocytes , elliptocytes )

69. Laboratory findings in intravascular hemolysis: a- Hemoglobinaemia b- Hemoglobinuria . c- Hemosidrinuria . d- Metheamalbuminaemia .

70. 1. Family history: It is an inherited disease 2. Full blood count: Hb concentration and PCV are low in patients with HbSS . 3. Blood morphology: sickle cells, polychromasia , anispoikilocytosis , and target cells are evident (See the below images) 4. Hb electrophoresis and cation -exchange-high performance liquid chromatography (CE-HPLC). See the below diagrams 4. Special tests: a. Sickling test: The sickling test is found to be positive in patients with Hb AS, and it is performed by a powerful reducing agent (sodium dithionate-Na 2 S 2 O 6 ). The test must be done in the deoxygenated condition, decreasing the solubility of Hb S and inducing the formation of sickle cells. b. Solubility test (See the below image) Laboratory diagnosis of sickle cell anaemia

71. In sickle cell disease Hb s > 80%, Hb F ( 1- 20% ), Absence Hb A. HB A 2 2- 4.5 %

72. Image demonstrates sickle solubility test. The Sickle Solubility Test (SST) is used to screen for the presence of sickling Hb. The SST utilizes a procedure based upon phosphate solubility whereby RBCs are lysed by saponin and the released Hb is reduced by sodium hydrosulfite in a phosphate buffer. Reduced HbS is characterized by its very low solubility and the formation of neumatic liquid crystals ( tactoids ). The resulting tactoids of HbS or non- sickling Hb (e.g. HbC -Harlem) causes the solution to remain turbid. The presence of HbA under these same conditions results in a clear red solution.    Hb A (clear red solution)   Hb S (Turbid red solution ) Solubility test

73. Figure demonstrates the abnormal Hb on CAM, alkaline pH Hb electrophoresis

74. A F A S SC SS Cation -exchange-high performance liquid chromatography

75. Hypochromic microcytosis blood picture can be caused by iron deficiency anaemia (IDA), thalassemia, lead poisoning, sideroblastic anaemia, or anaemia of chronic disease. How can you differentiate between these diseases and thalassaemia ? 1. Family history: The patient's history can exclude some of these etiologies . 2. Full blood count: - Low Hb and PCV. - The MCV is usually less than 75 fl with thalassaemia - The RDW will be elevated in more than 90% of persons with IDA and sideroblastic anaemia. , but in only 50% of persons with thalassaemia 3. Blood morphology: There is a slight imbalance in α / β chain synthesis, leading to hypochromic microcytosis poikilocytosis , and target cells. Laboratory diagnosis of thalassaemia

76. 4. Iron profile (serum Iron, Transferin saturation, TIBC and Ferritin), as discussed previously. 5. Hb electrophoresis or CE-HPLC, as shown in the last two images respectively. 6. Genetic analysis 7. BM examination for iron: BM aspirate, stained with Prussian blue or alizarin red dye d etects the iron in the BM, as shown below:

77. Diagnosis of β -Thalassemia Major Hypochromic microcytic anemia, nucleated red blood cells and anisocytosis . Hemoglobin electrophoresis in comparison with the normal control

78. The diagnosis of G-6-PD deficiency is suggested by : 1. Family history 2. Blood morphology: contracted and fragmented cells in the blood film. 3. By the demonstration of Heinz bodies. Reticulocytes have higher levels of G-6-PD than mature cells 3. Enzyme assay. Note Its results can be misleading during a haemolytic episode; thus it should be performed later. Laboratory diagnosis of G-6-PD deficiency

79. Pruvate kinase deficiency is inherited as autosomal recessive. The anaemia , which may be moderate to severe, cause relatively mild symptoms as there is a shift to the right in the O2 dissociation curve due to a rise in intracellular 2,3 DPG. Pyruvate kinase deficiency presents in childhood with mild jaundice. 1. Family history 2. Full blood count (Hb concentration and PCV will be low) 3. Reticulocyte count will be high because of haemolysis. 4. The diagnosis is also made by measuring erythrocyte pyruvate kinase activity Pruvate kinase deficiency Laboratory diagnosis of Pruvate kinase deficiency

80. WBCs disorders –Lab 4

81. Disorders of leucocytes may be quantitative or qualitative. Quantitative disorders are related to the concentration of leucocytes (leucocyte counts) in peripheral blood. Qualitative disorders refer to the structural or functional abnormalities of white blood cells. Leucocytosis is defined as an increase in the number of circulating leucocytes (total leucocyte count) above the upper level of normal. Leucopaenia refers to total leucocyte count below the lower limit of normal. An absolute rise or fall in the count can affect any white blood cell in peripheral blood, i.e. neutrophil, eosinophil, basophil, monocyte, or lymphocyte .

82. Leucoerythroblastic reaction refers to the presence of immature white blood cells as well as nucleated red cells in peripheral blood. Leukaemoid reaction refers to the presence of markedly increased leucocyte count (>50,000/ cmm ) and immature white blood cells in peripheral blood resembling leukaemia but occurring in non- leukaemic conditions.

83. Quantitative disorders of leucocytes

84. Shift to left

85. Shift to left

86. LEUKAEMOID REACTION: ‰‰‰‰ blood picture resembles chronic myeloid leukaemia . Marked neutrophilic leucocytosis with presence of premature white cells of all stages (from myeloblasts to segmented neutrophils) may mimick chronic myeloid leukaemia (CML).

87. DISORDERS OF PHAGOCYTIC LEUCOCYTES CHARACTERISED BY MORPHOLOGIC CHANGES: Acquired Morphologic Changes in Neutrophils Toxic granules Döhle inclusion bodies and cytoplasmic vacuoles that are seen in bacterial infections . Hypersegmentation of nuclei (>5 lobes in >5% neutrophils) is a characteristic feature of megaloblastic anaemia

88. Inherited Morphologic Changes Alder-Reilly Anomaly Characterized by the presence of abnormally large, darkly staining granules resembling toxic granules in cytoplasm. The granules are also variably present in monocytes. Commonly seen in mucopolysaccharidoses such as Hurler’s and Hunter’s syndrome.

89. Alder-Reilly Anomaly

90. Pelger -Hut Anomaly In this autosomal dominant disorder, nuclear segmentation does not occur in granulocytes. Granulocyte nuclei may be rod-like, round, or at the most with two segments (spectacle-like or “pince-nez” nuclei) . Survival and function of these granulocytes is normal

91. Pelger -Hut Anomaly

92. Shediak higashi syndrome

93. Acute leukaemia –Lab -5

94. Classification of leukaemia There are two types of acute leukaemias : a. Acute Lympho blastic Leukaemia (ALL) b. Acute Myelo blastic Leukaemia (A M L) or Acute Non Lympho blastic Leukaemia (A NL L). Diagnosis of acute Leukaemia FAB (French American British system) Classifications This system classifies patients with acute leukaemia according to: 1. Blood and bone marrow (BM) Morphology : appearence of cell under microscope. 2. Cytochemistry : chemical activity of the cell .( myeloperoxidase , Sudan Black B) 3. Immunophenotyping : antigen présent in the cell membrane, using flowcytoetry 4. Cytogenetics : Concerning with the study of the structure and function of the cell, especially the chromosomes.

95. 5. Genetic analysis : Identification of genes and inherited abnormalities FAB classification for Acute lymphoblatic leukaemia (ALL): ALL is classified into three categories based on the FAB classification system. ALL-L1 ALL-L2 ALL-L3 This classification according to the cellular morphology. L1 and L2 can be either B- or T-lineage, whereas L3 is only B lineage. ALL-L1:- It is associated with small homogenous lymphoblasts . Although the nucleocytoplasmic ratio is high , the scanty cytoplasm demonstrates variable basophilia . Approximately 80% of ALL cases are classified as L1 ALL-L2:- L2 is associated with much larger, heterogeneous. Although variable in size, the cytoplasm tends to be more abundant, therefore the nucleocytoplasmic ratio is lower .

96. approximately 18% of cases of ALL are classied as L2 categories ALL-3:- it constitutes about 2% of all cases ALL. It is essential that all cases of L3 are correctly claasified as this particular subtype is associated with specific therapeutic treatment Differential diagnosis of ALL and AML according to the cell morphology Morphology ALL ANLL (AML) Cell size small/moderate Moderate/large Nuclei usual 1 or 2 nucleoli Frequently more than2 nucleoli. Cytoplasm Scanty/moderate; homogeneous Moderate/abundant; granular, sometimes with Auer rods

97. Table shows differential diagnosis of ALL and AML according to the cytochemistry stain Cytochemistry stain ALL ANLL 1. Peroxidase Negative Positive 2. Sudan black Negative Positive 3. Acid phosphatase Positive Usually negative Differential diagnosis of ALL and AML according to the immunological markers Cluster differentiation (CD) ALL ANLL (AML_ CD19; CD22 Positive (B cell) Negative CD7, CD2 Positive (T cell) Negative CD13 and CD33 Negative Positive CD117 Negative Positive Glycophorin Negative Positive(M6) Key:- c= Cytoplasmic

98. FAB classification Morphological features Immunophenotype Membrane markers L1 Small; homogenous cell population Early B cell: null (n) cell ALL CD19 L2 Large; heterogenous cell population; one or two nucleoli Pre B cell: common (c) ALL T cell ALL D19 and CD10 2.CD2 and CD7 L3 Large; vaculated cytoplasm; heterogenous cell population; nucleoli prominent Mature B cell: ALL CD19 and sIg The FAB morphological classification of ALLwith corresponding immunophenotype Key:- sIg = Surface immunoglobulin

99. FAB Classification Comments FAB Classification M0 Undifferentiated myeloblast No cytochemical markers can be defined M1 Acute myeloblastic leukaemia without differentiation M2 Acute myelocytic leukaemia with differentiation Also on M2 basophil myeloblasts present. t(8; 21) M3 Acute promyelocytic leukaaemia Hypergranular promyelocytes . t(15; 17) M4 Acute myelomonocytic leukaemia Myeloid elements resemble M2, peripheral blood monocytosis . inv M5 Acute monocytic luekaemia M6 Acute erythroleukaemia M7 Acute megakaryocytic leukaemia Myelofibrosis may also occur Table shows The FAB morphological classification of ANLL (AML)

100. Cytogenetics In humans, there are two chromosomes that determine sex: the X and the Y chromosome. If you have an XX - you are female If you have an XY - you are male. Diploid vs Haploid -Body cells have the full set of chromosomes – they are -- DIPLOID (In humans, 46) HAPLOID (In humans, 23): Sex cells (sperm and eggs) have half a set (23). A karyotype shows all the chromosomes of an individual, in humans we see 22 pairs of autosomes, plus 1 pair of sex chromosomes. (total of chromosomes = 46)

101. Chromosomal abnormalities It is found in over 80% of cases of ANLL (AML) , the commonest abnormality being trisomy 8. Some translocations are associated with specific subtypes e.g : 1. t(15; 17) is unique to the M3 type 2. t (8; 21) is found in about 25% of M2 cases, usually young males. 3. Inversion of 16 or deletion of 16q occurs in about 25% of M4 cases and is associated with eosinophilia . Chromosomal abnormalities may also influence prognosis for instance : 1. t(8; 21) and inv 16 indicate a favourable diagnosis 2. t(15; 17) and del Y are of intermediate prognosis 3. Others carry a poor prognosis.

102. Flow cytometry Immunophenotyping by flow cytometry is an important tool in the diagnosis and staging of patients with a haematological neoplasm. It is used in conjunction with classical morphology. At each stage, the cells carry a distinctive set of markers classified by the CD (cluster of differentiation) nomenclature. Malignancies can arise at different stages in the development of a cell. A leukaemia or lymphoma will express a specific set of markers depending on the stage and pathway of differentiation and they are classified accordingly (see the below graphs ). B cell : CD5, CD10, CD19, CD20, CD45, Kappa, Lambda T cell : CD2, CD3, CD4, CD5, CD7, CD8, CD45, CD56; Myelomonocytic : CD7, CD11b, CD13, CD14, CD15, CD16, CD33, CD34, CD45, CD56, CD117, HLA-DR; Plasma cell : CD19, CD38, CD45, CD56, CD138 .

103. Chronic Leukaemia –Lab 6

104. Chronic Lymphocytic Leukaemia WBCs count: The outstanding feature is a marked increase in leucocytes, often 100,000/ cmm , or higher Blood picture: Nearly all are mature small lymphocytes See the below picture. Variants of Chronic Lymphocytic Leukaemia 1. Prolymphocytic leukaemia 2. Hairy cell leukaemia 3. T cell Chronic Lymphocytic Leukaemia CLL. The peripheral blood smear shows an absolute lymphocytosis of small “mature” lymphocytes with clumped, smudgy chromatin and scant cytoplasm. Smudge cells (near top right) are a frequent finding.

105. Diagnostic and course Minimum diagnostic criteria are: (1) a persistent circulating lymphocyte count of >5x10 3 / cmm (2) BM lymphocytosis > 30%. The clinical course is extremely variable: it may be rapidly progressive with a fatal outcome in 1 -2 years or it may be static over decades. Stage 0 Lyphmphocytosis Stage 1 Lyphmphocytosis and enlarged lymph nodes Stage II Lynphmphocytosis and enlarged liver or spleen or both, with or without enlarged nodes Stage III As with 0, I, II but Hb < 11 g/dl Stage IV As with 0, I, II or III but platelet count < 100, 000 / cmm Table shows Clinical staging of CLL

106. Chronic granulocytic leukaemia While commonest in adults of 30 – 40 years, this disease can occur at any age. The clinical picture is usually dominated by growth hepatic and splenic enlargement. Signs of impaired marrow function such as anaemia or thrombocytopenia are in conspicuous until late in the disease. After a variable period, usually several years, ANLL or common ALL supervene. It is generally accepted that CGL arises as a result of a mutation or series of mutation in a single pluripotential haemopoietic stem cell. Confirmatory evidence for the monoclonal origin of CGL comes from the distribution of the Philadelphia chromosome (Ph) in haemopoietic precursors of CGL patients Acute transformation in CGL may affect any or several of these lineages.

107. Philadelphia chromosome. A piece of chromosome 9 and a piece of chromosome 22 break off and trade places. The bcr-abl gene is formed on chromosome 22 where the piece of chromosome 9 attaches. The changed chromosome 22 is called the Philadelphia chromosome.

108. CML Phases The clinical courses of CML can be divided into three distinct phases:- a. Chronic phase b. Accelerated phase c. Blast crisis Chronic phase The majority of patients who present are in chronic phase, which may last between 2 and 7 Yrs Chronic phase is usua lly associated a hypercellular BM with peripheral leucocytosis and is responsive to therapy (good prognosis). The majority of patients at presentation have splenomegaly or hepatosplenomegaly and this can be reduced in chronic phase, using standard chemotherapeutic agents. Accelerated phase As the disease progresses to accelerated phase, it becomes more difficult to treat (bad prognosis).

109. The blast (immature) count will increase, although remains less than 20% and the basophil count will equal or exceed 20% ( basophilia ). The leucocytosis will become difficult to control and will accompanied with by either a persistent thrombocytopenia (less than 100x10 3 / cmm ) or a thrombocytosis (in some cases more than 1000 x10 3 /000/ cmm ) The presence of thrombocytopenia is part of disease process and is not secondary to cytoreductive therapy . Additional cytogenetic abnormalities may be found suggesting disease progression. Blast crisis describes the transformation to acute leukaemia The blast count is 20% or higher and immuophenotyping is required to determine the lineage of the acute leukaemia

110. Laboratory features The leukocyte count above 250000/ cmm (often above 100000/μ l ), granulocytes at all stages of development The basophiles count ( basophilia ) is increased The platelet count is normal or increased It is quite important to distinguish between a pronounced reactive leucocytosis ( leukaemoid reaction), as it is closely similar CML. Leukaemoid reactions are defined as leucoctosis associated with immaturity of the WBCs, so that myeloblasts and promyelocytes are seen in the peripheral blood. This WBC immaturity, which may resemble CGL. Leukaemoid reaction is due to causes such as viral infection, inflammatory reaction or neoplasia . The WBCs count in patients with Leukaemoid reaction may reach 50x 10 3 / cmm to 100x 10 3 / cmm with level in excess 100x 10 3 / cmm rarely reported

111. The diagnosis of CGL is usually easy, but a pronounced reactive leucocytosis ( leukaemoid reaction) can closely mimic CGL. Features which confirm the diagnosis of CGL include:- A high proportion of myelocytes Elevation of serum vitamin B12, probably due to an increase in plasma vitamin B12-binding protein. Low to absent levels of leucocyte alkaline phosphatase (LAP) in patients with CGL At least 85% of patients with CGL will have the Philadelphia Chromosome, while this chromosomal abnormality is absent in patients with a leukaemoid reaction An absolute elevation in proliferating marrow cells.

112. Image shows a small, hypolobated megakaryocyte ( center of field) in a BM aspirate, typically of CGL. Picture shows peripheral blood (MGG stain): marked leucocytosis with granulocyte left shift

113. Bleeding disorders-Lab - 7

114. Abnormal bleeding may result from: Vascular disorders (Inherited and acquired disorders) Platelets disorders which include: a. Qualitative disorders (Dysfunction platelets ) b. Quantitative disorders (thrombocytopenia) 3. Defective coagulation (coagulopathy) [Inherited and acquired disorders] Clinical differences between diseases of platelets, vessel wall and coagulation factors Finding Platelets/ vessel wall disorders Coagulation disorders Mucosal bleeding Common Rare Petechiae Common Rare Deep haematomas Minimal Characteristic Bleeding from skin cuts Persistent Minimal

115. Inherited Vascular disorders :- Hereditary haemorrhagic telangiectasia. (Dilatation in blood vessels) The exact causes of telangiectasia are unknow, it may be genetic, environmental, or a combination of both 2. Connective tissue disorders 3. Giant cavernous haemangioma Acquired Vascular disorders :- Simple easy bruising Senile purpura Purpura associated with infections. Note: Purpura  is a condition of red or purple discolored spots on the skin. Thrombocytopenia and defective platelet function They are associated with abnormal bleeding and characterised spontaneous skin purpura , mucosal haemorrhage and prolonged bleeding after trauma.

116. Causes of thrombocytopenia Increased consumption of platelets a. Immune (drug induced, heparin, autoimmune) b. Disseminated intravascular coagulation (DIC). C. Thrombotic thrombocytopenic purpura (TTP) 2. Failure of platelet production a. Selective megakarocyte depression(drugs, chemical, viruses) b. Part of BM failure ( Cytotoxic drugs, radiopathy , aplastic anaemia, leukaemia, HIV, lymphoma and carcinoma) 3. Abnormal distribution of platelets: Splenomegaly 4. Dilutional loss: It happens in massive transfusion of stored blood to bleeding patients

117. *Failure of platelet production is the most common thrombocytopenia. *Rarely, failure of platelet production is congenital as a result of mutation of the c-MPL thrombopoietin receptor, in association with absent radii, or in May- Hegglin Increased destruction of platelets : It is autoimmune (idiopathic) thrombocytopenic purpura (ITP): ITP is classified into, chronic and acute. *Chronic ITP: Its highest incidence occurs among women aged 15 – 50 years.

118. Coagulation factors deficiency Inherited coagulation disorders a. Haemophilia A (VIII) b. Haemophilia B ( Chrismus disease) (IX) c. Von Willibrand disease (VWF) Acquired coagulation disorders include a. Liver disease b. Vitamin K deficiency (II, VII, IX and X). They are called vitamin K dependant factors. c. Disseminated intravascular coagulation (DIC) d. Haemorrhagic disease of the newborn (sterile gut …….. No bacterial growth. No vitamin K dependant factors. (II, VII, IX and X)

119. Haemophilia A Deficiency of FV111 is referred to haemophilia A and it is inherited as X linked Laboratory diagnosis of haemophilia A a. Activated Partial Thromboplastin Time (APTT) is prolonged b. Factor VIII clotting assay is low c. Prothrombin time (PT) and bleeding time are normal

120. Classes of Haemophilia A based on FV111 activity Haemophilia B (Christmas disease) Haemophilia B Deficiency of 1X is referred to haemophilia B and it is also inherited as X linked , as haemophilia A Haemophilia B is called Chrismas disease , as it is named after the first boy described with it, Stephen Chrismas . Both diseases, haemophilia A and haemophilia B almost occur exclusively in males. Classified as Severe Moderate Mild FVIII (IU/dl) Quantity of FVIII < 1 IU/dl 1 - 5 IU/dl >5 IU/dl Age at presentation Infancy < 2Yrs >2 Yrs Bleeding symptoms and frequency Frequent spontaneous bleeds into joints, muscles and internal organs. Severe bleeding after trauma Much fewer spontaneous bleeds than patients with severe disease. Minor trauma can precipitate a bleed No spontaneous bleeds. Bleeding after significant trauma/surgery

121. Factor 1X is coded by a gene close to the gene which is coded for factor V111near the tip of the long arm of the X chromosome . Carrier detection and antenatal diagnosis is detected the same as haemophilia A. Factor 1X have a longer biological half life compared to the factor VIII. Laboratory diagnosis of haemophilia B: a. Activated Partial Thromboplastin Time (APTT) is prolonged b. Factor 1X clotting assay is low c. Prothrombin time (PT) is normal Haemophilia C (Rosenthal’s disease) Haemophilia C is due to a deficiency of FXI and it is an autosomal disorders. Rare

122. Von willibrand disease (VWD) VWD is inherited as autosomal dominant and it is due either a decreased level or abnormal function of Von Willibrand factor (VWF) as a result of a point mutation or major deletion. VWF activates platelets adhesion to damaged endothelium and it the carrier molecule for factor VIII, preventing it from premature destruction. There are three types of VWD, type 1, type 2 and type 3 Approximately 70% of patients with VWD have a quantitative deficiency of normal functioning VWF (type 1 ) and approximately 25% have a structural /functional defect (type 2 ). The remainder have severe VWD where VWF is absent ( type 3). -VWD type 1 is due to partial quantitative deficiency of VWF. -VWD type 3 represents a complete deficiency in VWF. - VWD- type 2 occurs as a result of functional abnormality in VWF

123. Laboratory diagnosis of VWD VWF levels are usually low Activated Partial Thromboplastin Time (APTT) may be prolonged Factor VIII clotting assay is often low Bleeding time can be prolonged The platelet count is normal except for type 2 VWD PT, TT and fibrinogen are unaffected

124. Congenital abnormalities of fibrinogen are divided into 2 types:- Type I, or quantitative abnormalities (afibrinogenemia and hypofibrinogenemia): result from mutations that affect plasma fibrinogen concentration inherited on both chromosomal alleles and are frequently associated with a bleeding diathesis but occasionally a thrombotic event.   Type II, or qualitative abnormalities ( dysfibrinogenemia and hypodysfibrinogenemia ). marked by functional abnormalities of fibrinogen who carry one abnormal allele that may result in either bleeding or thrombosis.  

125. Acquired coagualtion disorders Acquired coagualtion disorders are more common than inherited coagualtion disorder. The acquired coagualtion disorders are classified into: Disseminated intravascular coagulation (DIC). Deficiency of vitamin K-dependent factors Malabsorption of vitamin K (e.g. indandion , coumarins ) Haemorrhagic disease of the newborn (sterile gut…..No bacterial growth) Biliary obstruction Inhibition of coagulation Miscellaneous a. Diseases with M protein b. Massive transfusion syndrome c. Therapy with heparin, defibrinating agents or thrombolytic

126. Table shows acquired bleeding disorders Primary disorder Acquired cause of bleeding Sepsis, shock, obstetric calamities , trauma, surgery, some malignancies, transplant rejection, recreational drugs, snake bite. Acute DIC Some malignancies, chronic infections, chronic kidney disease Chronic DIC (less severe than acute DIC) Vitamin K deficiency Dietary deficiency or mal-absorption reduces synthesis of vitamin K dependent factors. Newborns are vitamin K deficient and can present with haemorrhagic disease of the newborn. Amyloidosis (extracellular protein deposition) Impaired platelet function, amyeloid binds FX causing plasma FX deficiency and can interfere with fibrin polymerization

127. Primary disorder Acquired cause of bleeding Liver disease Reduced synthesis of coagulation factors and thrombocytopenia Renal disease Ureamia and increases prostacyclin release impair platelet function Autoantibodies to coagulation factors or platelets Reduction in affected coagulation factor or platelet numbers Dilutional coagulopathy Massive transfusion of stored blood products and/or volume replacement with blood substitutes (i.e. they do not contain coagulation factors and live platelet) can reduce platelet numbers and concentrations of circulating coagulation factors. Drug therapy Anticoagualnt therapy Various drugs impair platelet function.

128. Laboratory investigation of a suspected bleeding disorder Diagnosis of a bleeding disorder is dependent on the presence of bleeding symptoms in the patient, yet compiling history is subjective. When there is clinical suspicion of a bleeding disorders, characterization of the presence and nature of any defect (s) starts with the performance by biomedical scientists of screening tests that will indicate the area (s) of haemostasis affected (See the blow Table) Area of haemostasis Tests Result determinants Platelet /vessels Platelet count Platelet film examination Platelet function test Bleeding time Platelet count Platelet morphology Platelet function; VWF Platelet function; Coagulation Prothrombin time (PT)     Activated partial thromboplastin time (APTT)   Thrombin time (TT)    Fibrinogen activity Levels of factors II, V, VII, X and fibrinogen + inhibitors   Levels of factors II, V, VIII, IX, X, XI, XII, PK, HMWK and fibrinogen Level of fibrinogen    Direct measurement of fibrinogen Fibrinolysis D-dimers *  D-dimer test helps in diagnosis patients with thrombosis Direct measurement of Fibrin degradation product ( FDPs)

129. Prothrombin time (PT) In this test the patient’s plasma will form a fibrin clot via the extrinsic pathway and common pathway and the time taken to clot is recorded as the PT. The patient’s PT, measured in seconds, is compared to a reference range which will vary depending on the analytical technique and type of thromboplastin used. An elevation of PT may indicate one or more of the following:- Deficiencies of factors II, V, VII, X or fibrinogen. Autoantibodies against the above clotting factors. Anticoagulant drugs affecting the production of vitamin K-dependent factors (i.e. Warfarin) Anticoagulant drugs directly affecting thrombin Liver disease DIC Activated partial thromboplastin time (APTT) In this test the patient’s plasma will form a fibrin clot via the intrinsic and common pathways

130. An elevation of APTT may indicate one or more of the following: Deficiencies of factors II, VIII, IX, XI, XII, PK, HMWK or fibrinogen Some subtypes of VWD (due to associated FVIII deficiency) Autoantibodies against the above clotting factors Anticoagulant drugs affecting the production of vitamin K dependent factors, although the APTT is less affected than the PT because there are more non-vitamin k dependent factors contributing to the clotting time. Anticoagulant therapy with heparin Anticoagulant drugs directly affecting thrombin Vitamin K deficiency Liver disease DIC

131. Result interpretation of PT and APTT Result Interpretation High PT and APTT Deficiency exists in the common pathway PT only elevated FVII deficiency APTT only elevated Deficiency exists in the intrinsic pathway Thrombin time The thrombin time (TT) is a simple test, whereby thrombin is added to patient plasma to directly convert fibrinogen to fibrin. The elevation of TT above the reference may indicate one or more of the following: Hypofibrinogenemia –acquired causes such as DIC are more commonly encountered than hereditary types Dysfibrinogenemia can be hereditary or acquired (i.e. liver disease) Afibrinogenemia. Anticoagulant drugs directly affecting thrombin Hypoalbunaemia , amyloidosis, paraproteins and elevated levels of fibrin/fibrinogen degradation products (FDP) can interfere with fibrin polymerization.