HEMA 2 MIDTERM
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| 🇬🇧 | 🇬🇧 |
| What is Flow Cytometry | Used to diagnose leukemia, to see the immunophenotypic features of Leukemia |
| Cheaper procedure to diagnose Leukemia | • Cytochemical Reaction |
| Classifications of Cytochemical Reactions | 1. Non-enzymatic Cytochemical stain 2. Enzymatic Cytochemical stain |
| What are the Enzymatic Reactions | Myeloperoxidase Chloroacetate Esterase Alpha naphthyl butyrate esterase Acid phosphatase Leukocyte alkaline phosphatase (LAP Score) |
| What is the Use of Myeloperoxidase | • Differentiate granulocyte and monocyte from lymphocyte |
| Peroxidase | Enzymes that catalyze the oxidation of substances by hydrogen peroxide |
| Myeloperoxidase Principle | When peroxidase (enzyme) is present, it will catalyze the oxidation of substances by hydrogen peroxide to form an insoluble reddish-brown precipitate. AH2 + H2O2 PEROXIDASE A + 2H2O |
| Myeloperoxidase Purpose | Differentiates acute myelogenous monocytic leukemia from ALL (Acute Lymphocytic Leukemia) - ALL- can be stained - AML- cannot be stained Recommended method for the demonstration for Auer rods compared to the Romanowsky stains |
| Myeloperoxidase Reagents | Ü Fixative: buffered formalin-acetone (30s) ü Incubation mixture (15 min) ü Counterstain: Giemsa (40 mins) |
| Myeloperoxidase Control | Positive: Neutrophils (because they contain granules with peroxidases, Brown in color if stained with Myeloperoxidase) Negative Lymphocytes (cannot be stained with Myeloperoxidase |
| Myeloperoxidase Cell specificity | Granulocytes Monocytes |
| Myeloperoxidase Interpretations | Positive (+): reddish brown/dark brown The RBC is also brown in color because they contain pseudoperoxidse activity |
| What is Chloroacetate Esterase | Used to differentiate granulocytes from monocytes Marker for mature and immature neutrophil/mass cells Less sensitive that peroxidase stain (in terms of primitive myeloid) |
| Chloroacetate Esterase Control | • Neutrophils |
| Chloroacetate Esterase Interpretation | • Positive: Bright red granules |
| What is Alpha naphthyl butyrate esterase | Butyrate esterases do not stain lymphoblast, plasma cell or megakaryocytes Useful in identifying monocytes, promonocytes, and monoblasts Can identify acute/ chronic myelomonocytic, acute monocytic, from other nonlymphocytic leukemia |
| Alpha naphthyl butyrate esterase Interpretation | Positive: dark red precipitate |
| Alpha naphthyl acetate esterase | Lymphocyte: show focal dotlike staining Monocytes: red-brown |
| What is Acid phosphatase | Present in all hematopoietic cells and is found in lysosomes. This provides a simple test for identification of T-acute lymphocytic leukemia but should be evaluated with peroxidase and esterase |
| Acid phosphatase Interpretation | Positive: discrete purplish to dark red granules |
| Example of a test that uses Acid phosphatase | Tartrate Resistant Acid Phosphatase (TRAP)- helpful in diagnosing Hairy Cell Leukemia also known as Leukemic Reticuloendotheliosis |
| What Leukocyte alkaline phosphatase (LAP Score) | Is also known as neutrophil alkaline phosphatase because only neutrophils are the only leukocyte that normally contains various alkaline phosphatase To differentiate Chronic myelocytic leukemia (CML) from leukemoid reactions or other myeloproliferative disorders (PV, ET, PMF) The CML has a decrease Lap score compared to the Leukemoid reactions. |
| Lap score principle | Alkaline phosphatase activity is present in varying degrees in the neutrophil and band form of the granulocytes The amount of dye precipitated is proportional to the amount of enzyme present. Generally, a high WBC with increased neutrophils is associated with increased enzyme. Relationship between the number of enzyme and the Neutrophil present is Directly proportional With the use of different substrate solution with the presence of Enzyme Alkaline Phosphatase in an alkaline ph, it will be hydrolyzed. Using working solution (ex. Fast Blue RR dye or Fast Blue BBN) it will result to an insoluble precipitate at the site of enzyme activity. |
| Lap score reagents | • Fixative: formalin and methanol • Substrate solution: naphthol • Working solution: Fast blue BBN • Counterstain: nuclear fast red |
| Lap score Procedure | • Make a blood film or smears • Immerse blood films in cold (4–10-degree Celsius fixative for 30 secs) • Wash well in either tap or distilled water • Immerse fixed slides in working solution for 20 mins at room temperature • Wash well in tap water after fixing with the working solution. |
| Lap score control | • Blood drawn from a woman in her third semester of pregnancy or within 2 days postpartum • Best in their last trimester provides excellent positive controls. Above normal results |
| Lap score grading | • At least 2 patients’ slide are obtained with 2 observers and should count 50 neutrophils scoring from 0 to 4+ on the basis of the quantity and intensity of the precipitated red dye in their cytoplasm. no granules very few granules (faint, diffuse staining) moderate granules scattered numerous granules cytoplasm is packed |
| Lap score Normal | Normal LAP: 40-100 (steininger) Normal LAP: 30-185 (brown) The sum of the ratings of cells is the LAP score; the cell ratings and total score are reported |
| LAP elevated in” | Leukemoid reaction Pregnancy Polycythemia vera Aplastic anemia Multiple myeloma Obstructive jaundice Hodgkin’s’ disease |
| LAP decreased in: | CML Paroxysmal nocturnal Hemoglobinuria Sickle cell anemia Hypophosphatasia |
| What are the Non-enzymatic Reactions | Sudan Black B Periodic Acid Schiff Periodic Acid Schiff |
| What is Sudan Black B | Stains various lipids such as sterols, phospholipids and neutral fats Believed to be more sensitive than myeloperoxidase Differentiate acute myelogenous/ monocytic leukemia from ALL |
| SBB Reagents | Reagents: Fixative: formalin-acetone Sudan black B solution 70% ethanol Counterstain: nuclear fast red |
| SBB Procedure | Fixair-dried films in cold buffered formalin-acetone fixative for 30 to 60 seconds Washin running water for 1 minute Immersein working stain for 1 hour Rinsein70% ethanol for 2 minutes Washin distilled water for 2 minutes Counterstainwith nuclear fast red 10 to 15 minutes Wash,fan dry and apply coverslip with glycerol mounting medium for examination |
| Periodic Acid Schiff | Carbohydrates present in blood cells are oxidized to glycogen by periodic acid. Schiff’s reagent then reacts with the aldehydes to forms an insoluble red colored precipitate The pattern of reactions is diffuse, granular or a mixture of the two (ALL with positive PAS) |
| PAS interpretation | Positive bright fuchsia pink Neutrophils: diffusely and finely granular Basophils: positive Megakaryocytes are diffusely granular with large peripheral granules Monocytes: faintly positive Lymphocytes, erythrocytes precursors and eosinophil are negative |
| Toluidine Blue | Dye that can bind with acid mucopolysaccharides in blood cells to form metachromatic complexes (Alder reilly) Most useful in recognition of mast cell disease and acute/ chronic basophilic leukemias |
| Toluidine reagents | • Mota’s fixative (1 min) • 0.1% toluidine blue (2 mins) |
| Toluidine Control | • Positive: buffy coat preparations of normal peripheral blood |
| Toluidine Interpretation | • Nuclei will stain light blue metachromatic granules will appear reddish violet |
| Neutrophil Reduction Test | Nitroblue tetrazolium test (NBT) |
| Nitroblue tetrazolium test (NBT) | Done to screen for CGD (Chronic Granulomatous Disease- lack of respiratory bust, not capable of killing microorganism) |
| NBT principle | A yellow water-soluble dye, NBT on reduction is converted to an insoluble blue formazan |
| NBT Procedure | 0.1 mL of blood is mixed with 0.1 mL of 0.2% nitro-blue tetrazolium is saline and 0.1 mL of phosphate buffered saline. This mixture is incubated at 37°C for 15 minutes and then held at room temperature for 15 mins. |
| Platelets | Ü Aka thrombocytes ü Platelets are notactually cells, rather they are cytoplasmic fragments of megakaryocytes-(progenitor or shredding of platelets) |
| Platelet’s maturation series | Stem 1. Multipotent hematopoietic stem cell Progenitor 2. Common myeloid progenitor 3. Megakaryocyte-erythrocyte progenitor Precursor 4. Megakaroblast Mk-I 5. Promegakaryocyte Mk-II 6. Megakayocyte Mk-III Peripheral Blood 7. Platelets |
| Platelet appearance | On a PBS, they appear as bluish, round to oval, anucleated, granular structures that are 2 to 4 um in diameter (Barbara- 1 to 4 um) 2/3 of platelets can be seen in the blood stream, 1/3 of it is sequestered in the spleen. |
| Level of platelets on patients with splenectomy and splenomegaly | If the patient has splenectomy, the platelet count will increase. And splenomegaly can cause decrease of platelets. |
| Life span of platelets | 8 to 11 days is the average life span of platelets. |
| Primary function | For sealing, hemostasis They form the 2nd major component of the hemostatic system Note: The 1st major component for the maintenance of Hemostatic system are the Blood vessels. (Contains the cell if there is wound, and the platelets will seal the site of injury or wound) They have sealing function – they aid in the formation of thrombus, or plug (Platelet Plug Formation to stop the breakage in the endothelial system) when there is a break in the circulatory system. |
| Significant decrease in the number of platelets | Leads to gaps in vessels, resulting to leakage of blood into tissues |
| Normal concentration of platelets: | • 150,000 – 450,000 /uL (150-450x109/L) |
| Manual (Direct) | 1. Reese and Ecker • (Sodium citrate, formaldehyde, Bromcresol blue) - reagent • Light microscope 2. Guy and Leake • Sodium oxalate, Formaldehyde, Crystal violet • Light Microscope 3. Brecker-Cronkite- Reference method • Brecker-Cronkite • 1% ammonium oxalate- reagent • Phase contrast microscopy 4. Ubopette • 1% ammonium oxalate - reagent • Phase contrast microscopy |
| Indirect | • Dame check • Fonios • Olef |
| Automated | 1. Electronic particle counter - Same counting in the RBC using electronic particle counter. - If there is wavelength in the cells, the counter will measure and check the size. If smaller than the RBC it is counted as platelet. - Prone to error (the rouleaux cell might be counted as one) |
| Platelet Estimate Purpose | • To provide a rapid approximation (estimate) of platelet numbers; and • To verify (cross-check) the result of an instrument platelet count |
| • Mistaken with | Debris, refractile or granular debris • Adhere to blast and fellow platelets and produce erroneous results because in the automated counting it can be counted as one |
| Thrombocytosis | • Increased platelet count (chronic myelocytic leukemia, polycythemia vera, sickle cell anemia) |
| Thrombocytopenia | • Decreased platelet count (pernicious anemia, dengue fever, acute leukemia) |
| Platelets are stored in | In room temperature with constant agitation. |
| For the estimate to be valid, platelets should: | A. Not be clumped b. Be observed in the area where RBCs do not overlap |
| In a properly prepared smear | A. Each oil-immersion field should yield between 8-20 platelets |
| Action | • Have blood sample redrawn w/ Sodium citrate |
| Materials | 1. Capillary blood sample (less preferred) or venous blood sample – EDTA (more preferred) 2. Slides and / or spreader 3. Wright stain 4. Microscope 5. Immersion oil |
| Capillary blood vs. Venous Blood | 1. Capillary blood sample (less preferred because after collecting blood sample, you need to immediately perform smear and the platelets are prone to clumping and also the adhesion of platelets in the injured vessel causing False Decrease) or venous blood sample – EDTA (more preferred, evenly distributed and can be performed later) |
| Regions in counting | - Count in the C region because there are no rouleaux formation - Region A, B, C |
| Methods | 1. Obtain blood sample 2. Make a smear. Stain using Wright stain 3. With the aid of the immersion oil, examine the dried Wright-stained smear under OIO 4. Count the number of platelets seen per OIO. View10 fields 5. Compute platelet estimate using formula |
| Platelet estimate / uL = | Platelet estimate / uL = average # of platelets counted x 20,000 (factor) Platelet estimate /uL = # of platelets x area (1) x depth (10) x dilution (200) |
| REFERENCE RANGE | Marked decrease Moderate decrease Slightly decreased Low normal Normal Slightly increased Moderate increase Marked increase |
| 30, 000 to 50,000 | Bleeding possible with trauma |
| >30,000 | Spontaneous bleeding |
| >5000 | Severe spontaneous bleeding |