Hematology EDTA (Lavender top)
Modified Westergren ESR (Black top tube) Citrate (Light blue top tube)
Polycythemic patients
Oxalate Heparin
Order of Draw (Henry 21st Edition)
Order of Draw (Syringe method)
MUST TO KNOW IN HEMATOLOGY Greek: -Haima = Blood -Logos = Study/science Chelates calcium Inversion: 8x Anticoagulant of choice for hematology cell counts and cell morphology Blood smear: prepare w/in 2 hrs Preferred anticoagulant for platelet count: = In some patients w/ EDTA anticoagulated blood – platelet satellitism = Platelet satellitism: platelets adhere to neutrophils ♫ Effect to automated platelet count Decreased ♫ Remedy: Repeat platelet count using citrate (Rodak: Platelet count x 1.1) EDTA = Shrinkage of cells = Hct = ESR Not for coagulation tests: = Inhibits fibrinogen-thrombin reaction = Factor V is not stable in EDTA 2mL EDTA + 0.5mL NSS/Citrate Ratio = 1:4 (Anticoagulant-to-Blood) For coagulation and platelet studies = Preserves labile factors V and VIII = Buffered 3.2% (0.109M) citrate Inversion: 3-4x Ratio = 1:9 (Anticoagulant-to-Blood) Hct Excess Citrate = PT, APTT Remedy: Reduce the volume of citrate Amount of citrate = [(100-Hct)÷(595-Hct)] x mL WB Double/balanced oxalate (Ratio = 2:3): Maintained cell structures a. Potassium oxalate (Paul-Heller’s) = shrink cells b. Ammonium oxalate (Wintrobe’s) = swell cells Inactivation of thrombin Anticoagulant for osmotic fragility test Inversion: 3-4x Not for blood film preparation: = Distorts cells = Produces bluish background on Romanowsky’s stain Not for coagulation = Inhibits thrombin and all stages of coagulation Evacuated tube: 1. Sterile blood culture tube 2. Citrate (blue) 3. Nonadditive tube (red) 4. Heparin (green) 5. EDTA (lavender) 6. Fluoride (gray) 1. EDTA 2. Other anticoagulated tubes 3. Nonadditive tube Page | 177
EDTA containing tubes
Skin puncture
Venipuncture
Common gauge (needle) Common length of needle Color coded hub (gauge)
Angle Tourniquet BP cuff as tourniquet Reassure the patient Position the patient IV line
Hematopoiesis Mesoblastic period
Hepatic period
Lavender Pink White Royal blue Tan 1. Fingertips 2. Earlobe: less admixture w/ tissue juice, less pain, less free nerve endings 3. Lateral portion of the plantar surface of the foot: <1 year old Difference from venous specimen: WBCs Hgb, Hct, RBCs, platelet Veins in the arms (antecubital region): 1. Median cubital = preferred, most stable 2. Cephalic (lateral) 3. Basilic (medial) 19, 20, 21 Routine: 20g 1-1.5 inches 18 = pink 21 = green 22 = gray 23 = blue/light blue/turquoise Venipuncture: 150 BB: 450 10-200 once in the skin 3-4 inches above the site (7.5-10cm) Not exceed 1min/2mins Prolonged application hemoconcentration 40-60 mmHg Crying = cell count Lying down = hemodilution ( PCV by 8%, WBC) Lying up = hemoconcentration Collect on the other arm If both arms: Stop IV for 2mins = Collect blood below the IV line = Appropriate for all analytes except glucose and phosphorus Cellular formation, proliferation, differentiation and maturation of blood cells 19th day of gestation Yolk salk = Erythropoiesis Embryonic hemoglobins: a. Gower 1 = Zeta2 + Epsilon2 b. Portland = Zeta2 + Gamma2 c. Gower 2 = Alpha2 + Epsilon2 3rd month of gestation Fetal liver = Granulopoiesis, Erythropoiesis, Megakaryopoiesis Spleen, thymus, lymph nodes Hemoglobin production: a. HbF = Alpha2 + Gamma2 b. HbA1 = Alpha2 + Beta2 c. HbA2 = Alpha2 + Gamma2 Page | 178
Myeloid period Adults Neonates Marrow specimens
Posterior iliac creast M:E ratio
BM Cellularity
Yellow BM Red BM Marrow differential Metamyelocyte/Juvenile granulocyte Stem cells Osteoblasts Osteoclasts CD2, CD3 CD19, CD20 CD34 CD16, CD56 CD10 Erythropoietin Thrombopoietin Erythropoiesis IL-3
Between 5th & 6th month of gestation persist throughout life BM = 1’ source of cell production (Hematopoiesis) Sternum = principal source of hematopoiesis in adults HbA1 = ≥95% HbA2 = 1.5-3% HbF = <2% HbF = 60-80% HbA = 20-40% 1. Trephine (Core) Biopsy = Trephine biopsy needle (Jamshidi needle) 2. Aspiration = Aspiration needle (University of Illinois sterna needle) Safest site for BM aspirate/biopsy Numeric expression comparing the relative number of granulocytic precursors w/ the relative erythroid precursors in the BM NV = 2:1 to 4:1 (Ave. 3:1) Infection = 6:1 Leukemia = 25:1 Neutrophilic, Eosinophilic, Basophilic precursors = Myeloid Erythroid precursors Monocytic precursors = not included Percentage of marrow space occupied by hematopoietic cells compared w/ fat Normocellular marrow (Adult): ♫ Fat = 10-50% ♫ Hematopoietic elements = 40-60% (Ave. 50%) Fats Hematopoietic cells Recommended that at least 500, preferably 1000 cells be counted for a marrow differential Predominant cell (WBC) in adult BM (up to 32%) <1% cells in BM Bone forming cells Confused w/ plasma cells Waterbug or comet appearance Bone destroying cells Confused w/ megakaryocytes T cells B cells Stem cell marker (lymphoid and myeloid precursor) NK cells CALLA (Common ALL Antigen) Produced by the kidney Primary regulator of erythropoiesis Produced by kidney and liver Regulator of thrombopoiesis 1’ stimulus = Hypoxia Multi-CSF (Colony Stimulating Factor) Page | 179
1. Pronormoblast
2. Basophilic normoblast
Stimulates hematopoietic cells Proerythroblast/rubriblast N/C ratio = 8:1 Nucleoli = 1-2 Can produce up to 16 RBCs per 1 pronormoblast/rubriblast Prorubricyte Intensely basophilic cytoplasm N/C ratio = 6:1 Nucleoli usually not visible
3. Polychromatophilic normoblast
Rubricyte Blue-gray to pink-gray cytoplasm Last stage capable of mitosis 1st: Hgb synthesis (1st: PCPNB Reticulocyte: Last) N/C ratio = 4:1 4. Orthochromic Metarubricyte normoblast Small pyknotic nucleus (dark, small, nonfunctional) N/C ratio = 1:2 5. Reticulocyte Polychromatophilic erythrocyte/Diffusely basophilic erythrocyte Romanowsky stain = Polychromasia Supravital stain = (+) Fine reticulum of RNA 6. Mature RBC Discocyte 6-8 μm in diameter Life span: 120 days 3-5 days BM: Pronormoblast Reticulocyte 1-2 days PB: Reticulocyte RBC General Cell Maturation Characteristics for Leukocytes Immature Cells Mature Cells Larger Smaller (+) Nucleoli (-) Nucleoli Chromatin: fine and delicate (most reliable) Chromatin: coarse and clumped (most reliable) Nucleus: large and round Nucleus: round. lobulated or segmented Cytoplasm: dark blue/basophilic (RNA) Cytoplasm: light blue (RNA) (-) Granules (+) Granules N:C ratio N:C ratio Granulopoiesis Neutrophils Eosinophils Basophils 14 days Blast Mature granulocyte 1. Myeloblast Earliest recognizable stage in granulocytic series N/C ratio = 4:1 Nucleoli = 2-5 2. Promyelocyte 1st: Primary granules N/C ratio = 2:1 to 3:1 Nucleoli = 2-3 3. Myelocyte Youngest cell in the series wherein a granulocyte can be identified a. Neutrophil myelocyte = rose pink granules b. Eosinophil myelocyte = orange-red granules Page | 180
4. Metamyelocyte
5. Band
6a. Segmented neutrophil
6b. Eosinophil
6c. Basophil
Monopoiesis
c. Basophil myelocyte = dark purple or blue-black granules Last stage capable of mitosis 1st: Secondary granules N/C ratio = 1:1 (-) Nucleoli Juvenile granulocyte Not capable of mitosis (post-mitotic pool) Indented/kidney-shaped nucleus Predominant WBC in BM Stab/Staff Youngest cell in the series present in the peripheral blood (normal) PB = 0-6% or 0-7% C or S shaped nucleus Curved or Sausage shaped nucleus Resembles Pelger-Huet cells = PH cells: coarser chromatin than stab cells Rose-pink granules Nucleus: 2-5 lobes Diurnal variation (PM) Specific granules: a. Lysozyme b. Lactoferrin c. Collagenase d. Plasminogen activator e. Aminopeptidase Reddish-orange granules Nucleus: usually 2 lobes Diurnal variation (ACTH) Specific granules: [Larger] a. Major basic protein b. Acid hydrolase c. Cathepsin d. Eosinophil cationic protein d. Eosinophil-derived neurotoxin e. Eosinophil protein X f. Phospholipase [Smaller] a. Arylsulfatase b. Acid phosphatase Dark purple to blue-black granules (water-soluble) Nucleus: generally unsegmented or bilobed (rare: 3 or 4) Specific granules: a. Histamine b. Heparin c. Eosinophilic chemotactic factor A 1. Monoblast 2. Promonocyte 3. Monocyte Page | 181
Monocyte
Lymphopoiesis Lymphocyte
T lymphocytes B lymphocytes Null lymphocytes Plasma cells differentiation Plasma cell
Thrombopoiesis 1. Megakaryoblast 2. Promegakaryocyte 3. Granular megakaryocyte 4. Mature megakaryocyte
5. Metamegakaryocyte 6. Platelet/Thrombocyte
2/3 (67%) 1/3 (33%) Endomitosis
Largest cell in PB 14-20 μm in diameter Blue-gray cytoplasm Many azurophilic granules (ground glass appearance) Nucleus: kidney/horse-shoe shaped, may be folded (brainlike) 1. Lymphoblast 2. Prolymphocyte 3. Lymphocyte Small = 8-10μm (Size = RBC) Medium = 10-12μm Large = 12-16μm (Rare) Cytoplasm: bluish (Robin’s egg blue) Nucleus: compact 60-80% Long-lived (4-10 years) 10-20% Short-lived (3-4 days) Can differentiate into plasma cell or memory B cells Large granular lymphocyte 10% 1. Plasmablast 2. Proplasmacyte 3. Plasmacyte/Plasma cell Large well-defined hof/perinuclear halo (light staining area in the cytoplasm near the nucleus) Eccentric nucleus Deeply basophilic cytoplasm (Red/pink cytoplasm: Flame cell [Abnormal]) Chromatin: “Cart wheel pattern” 5 days (Megakaryoblast Platelets) N/C ratio = 10:1 Nucleus: may show slight lobulation (Endomitosis) N/C ratio = 4:1 to 7:1 Largest cell in BM Cytoplasm contains coarse clumps of granules aggregating into little bundles, which bud off from the periphery to become platelets Multiple nuclei N/C ratio = <1:1 Disintegrated cell surrounded by platelet 1-4μm in diameter Light blue to purple Very granular a. Chromomere: granular and centrally located b. Hyalomere: surrounds the chromomere, nongranular and clear to light blue Life span: 8-11 days Circulating platelets Platelets stored in the spleen Nuclear division w/o cytoplasmic division Page | 182
2000-4000 1 heme molecule 1 hemoglobin Mitochondria 141 amino acids 146 amino acids Chromosome 11 Chromosome 16 Oxyhemoglobin Dissociation Curve Shift to the left (ODC)
Shift to the right (ODC)
Heme synthesis
# of platelets a megakaryocyte can produce 1 mol O2 4 mol O2 Early and late heme synthesis Alpha Beta, Gamma, Delta, Epsilon, Zeta Alpha, Zeta Beta, Gamma, Delta, Epsilon Normal = Sigmoid in shape X-axis = Hgb concentration in g/dL | Y-axis = OD CO2 Temperature 2,3-DPG pH Affinity of Hgb for O2 HbF CO2 Temperature 2,3-DPG pH Affinity of Hgb for O2 Succinyl coenzyme A + Glycine + Pyridoxal PO4 ↓ (ALA synthase) ↓ D-Aminolevulinic acid ↓ (ALA dehydrase) ↓ Porphobilinogen ↓ (Uroporphyrinogen synthase) (Uroporphyrinogen cosynthase) ↓ Uroporphyrinogen ↓ (Uroporphyrinogen decarboxylase) ↓ Coproporphyrinogen ↓ (Coproporphyrinogen oxidase) ↓ Protoporphyrinogen ↓ (Protoporphyrinogen oxidase) ↓ Protoporphyrin IX + Fe2+ ↓ Page | 183
Arterial blood Venous blood P50 Bohr effect Oxyhemoglobin (HbO2) Deoxyhemoglobin (HbCO2) Carboxyhemoglobin (HbCO)
Methemoglobin/ Hemiglobin (Hi)
Sulfhemoglobin
Erythrocyte membrane
Embden-Meyerhoff pathway
(Ferrocheletase) ↓ HEME
O2 saturation = 95% pO2 = 95 mmHg O2 saturation = 70% pO2 = 40 mmHg pO2 = 26.6 mmHg pH = Hgb affinity for O2 --- (L) pH = Hgb affinity for O2 --- (R) Arterial blood Blood: Bright red color Venous blood Blood: Purplish red color Blood: Cherry red color Reversible Cannot bind and carry O2 CO has 200x or 210x greater affinity for Hgb compared to O2 Smoking, gas, etc. Blood: Chocolate brown color Reversible Fe2+ Fe3+ Methemoglobin reductase deficiency Exposure to chemicals/drugs Exposure to nitrite, chlorine and nitrate HbM (Abnormal hemoglobin) Blood: Mauve lavender Irreversible Cannot transport O2 Can combine w/ CO forming carboxysulfhemoglobin Mixture of oxidized, partially denatured forms of hemoglobin that form during oxidative hemolysis green hemochrome Sulfonamides, aromatic amine drugs Bacteremia: Clostridium perfringens Enterogenous cyanosis 50% protein: 1. Integral protein: Glycophorin A = Contains sialic acid: (-) charge = Zeta potential: red cells repel each other 2. Peripheral protein: Spectrin and Actin = Responsible for biconcave shape of red cells = Abn. Spectrin: Hereditary Spherocytosis, Hereditary Elliptocytosis 40% lipid: 1. Phospholipids 2. Cholesterol = LCAT 10% CHO Major pathway 90% glycolysis (anaerobic) Glucose Lactic acid = 2 ATP Page | 184
Hexose monophosphate shunt (Pentose PO4 pathway) Rapoport-Luebering pathway Methemoglobin reductase pathway Pitting Culling RBC lysis Extravascular hemolysis
Intravascular hemolysis
Anisocytosis RDW Anisochromia MCV MCH MCHC Normocytic normochromic anemia Microcytic hypochromic anemia
Macrocytic, normochromic anemia Aplastic anemia
PK deficiency: common enzyme deficiency 10% glycolysis (aerobic) Provides reduced glutathione to prevent Hgb denaturation G6PD deficiency: most common red cell enzyme deficiency = (+) Heinz bodies: denatured Hgb (Supravital stain: RHH) = (+) Bite cells: due to pitting Generates 2,3-DPG that regulates Hgb affinity for O2 Maintains Hgb iron in ferrous (Fe2+) state to be functional Removal of inclusion by the spleen Removal of senescent/aged RBCs by the spleen RBC age = Enzymes, ATP, Size, Density 1% of RBCs/day broken down by the mononuclear phagocytic system (MPS) 90% aged red cell destruction w/in the RES when C’ is not/incompletely activated Unconjugated bilirubin Urine and fecal urobilinogen 10% aged red cell destruction w/in the blood vessels when C’ is completely activated Haptoglobin Hemopexin (+) Hemoglobinemia Variation in size Numerical expression that correlates w/ the degree of anisocytosis NV = 11.5-14.5% Variation in Hgb content (Ex. Dimorphic anemia) NV = 80-100fL (Normocytic) <80fL = Microcytic >100fL = Macrocytic NV = 27-32 pg NV = 31-36% (Normochromic) <31% = Hypochromic >36% = Hyperchromic 1. Acute blood loss 2. Hemolytic anemia 3. Aplastic anemia 1. Chronic blood loss = most common cause 2. Thalassemia = N-RDW 3. IDA = Iron, TIBC (Ex. Hookworm infections) | RDW 4. Sideroblastic anemia 5. Chronic disease 1. Megaloblastic anemia = Vitamin B12/Folate deficiency 2. Nonmegaloblastic anemia = Liver disease, alcoholism Congenital = Fanconi’s anemia Acquired = radiation, chemical (benzene), drugs (chloramphenicol) Page | 185
TIBC Degree of Hypochromia
Megaloblastic anemia
Vitamin B12 (Cobalamin) deficiency
Folic acid (Vit. B9) deficiency Polychromasia
Spherocytosis Microcytic, hypochromic Poikilocytosis Macrocytes Oval macrocytes Macroovalocytes Acanthocyte Spur cell Thorn cell Echinocyte Burr cell Sea urchin cell Crenated RBCs
Codocyte
Pancytopenia = WBCs, RBCs, Retics Plts Differentiates IDA (TIBC) from other microcytic, hypochromic anemia Normal = Area of palor 1/3 of the cell diameter 1+ = Area of palor 1/2 of the cell diameter 2+ = Area of palor 2/3 of the cell diameter 3+ = Area of palor 3/4 of the cell diameter 4+ = Thin rim of hemoglobin Oval macrocytes Howell-Jolly bodies Hypersegmented neutrophils Ineffective erythropoiesis (pancytopenia) ♫ w/ CNS problems 1. Pernicious anemia = Deficient in intrinsic factor (produced by parietal cells) for B 12 absorption 2. D. latum infection 3. Vegetarian diet 4. Malabsorption syndrome = Steatorrhea, sprue ♫ w/o CNS problems 1. Pregnancy 2. Dietary deficiency 3. Steatorrhea, sprue Reticulocytosis Visible on Wright’s stain Blue-gray coloration, pink cytoplasm Indicates young RBCs Erythropoietic activity a. Hemorrhage b. Hemolysis EOFT EOFT Variation in shape ESR = No rouleaux formation Megalocytes: Vit. B12 or folic acid deficiency Asynchronous development: mature cytoplasm but immature nucleus Irregular spikes/spicules Abnormal L:S ratio ♫ Abetalipoproteinemia Liver diseases Echinocyte: artifactual Burr cell: pathologic RBCs w/ projections of equal length and distribution ATP ♫ Exposure to hypertonic solution ♫ Artifact in air drying ♫ Anemia associated w/ renal insufficiency Greek: Kodon = bell Page | 186
Target cell Mexican hat cell
Leptocyte Spherocyte Ball Bronze cell
AIHA vs. HS Stomatocyte
Elliptocyte
Ovalocyte
Schistocyte Schizocyte Fragmentocyte
Keratocyte Knizocytes Pinch cells Dacryocyte
Central hemoglobinized area (bull’s eye) Scanning EM: Bell/tall hat shaped Surface membrane to volume ratio Cholesterol & phospholipid ♫ Hemoglobinopathies ♫ Thalassemia ♫ Liver disease, postsplenectomy, IDA Thin variant of a codocyte Spheroid Biconvex Surface area to volume ratio Defect of loss of membrane: ♫ Hereditary spherocytosis = Post-splenectomy: Spherocytes ♫ Hemolytic anemia ♫ Burns ♫ Banked blood stored for a long time AIHA = OFT, MCHC, (+) DAT HS = OFT, MCHC, (-) DAT Greek: Stoma = mouth Mouth, slit-like pallor area Bowl-shaped Permeability to Na+ (Normal: permeability to K+) ♫ Hereditary stomatocytosis ♫ Rhnull disease Rod/cigar shaped Narrower than ovalocytes Defect in cytoskeleton Membrane protein band 4.1 Egglike/oval-shaped Wider than elliptocytes Bipolar arrangement in Hgb Cholesterol ♫ Hereditary ovalocytosis ♫ Megaloblastic BM ♫ Myelodysplasia
Fragmentation produced by damage of RBC by fibrin, altered vessel walls, prosthetic heart valves ♫ Microangiopathic hemolytic anemia = Presence of fibrin strands in small blood vessels “clothesline-like effect” ♫ Burns ♫ TTP ♫ DIC Schistocyte w/ hornlike projections Triangular cells 2 palor areas Teardrop/pear-shaped Page | 187
Teardrops Microspherocyte Pyropoikilocyte
Semilunar bodies Half-moon cell Crescent cell Burns Drepanocytes Sickle cells Menisocytes Howell-Jolly bodies Basophilic stippling Punctate basophilia PICA Cabot rings Heinz bodies
HbH inclusions HbCC crystals
HbSC crystals Ringed Sideroblast
Squeezing and fragmentation during splenic passage ♫ MMM ♫ Hypersplenism ♫ Severe burns ♫ Hereditary microspherocytosis ♫ Hereditary pyropoikilocytosis = sensitivity to temperature = RBCs fragment at 45’C (Normal: 49’C) Large, pale pink staining ghost of the red cell Membrane remaining after the contents have been released ♫ Malaria Spherocytes Schistocytes Microspherocytes Crescent-shaped cell Holly-leaf Polymerization of deoxygenated Hgb ♫ Sickle cell anemia ♫ Sickle cell disease Nuclear remnants of DNA (from karyorrhexis) Feulgen (+) ♫ Megaloblastic anemia Precipitation of ribosomes and RNA 1. Fine stippling = polychromatophilia ( production of RBCs) 2. Course stippling = Lead poisoning ♫ Pyrimidine-5-nucleotidase deficiency = Basophilic stippling In children = Lead poisoning In adults = IDA Figure of 8 Remnant of microtubules of mitotic spindle ♫ Megaloblastic anemia Precipitated, denatured Hgb Multiple Heinz bodies Pitted golf ball appearance Requires Supravital stain (RHH) ♫ G6PD deficiency ♫ Unstable hemoglobins (HbH) ♫ Favism (Fava beans) ♫ Drug-induced Acetylphenylhydrazine/phenylhydrazine = Induce Heinz bodies formation Tetramer of beta globin chains (β4) Requires Supravital stain (RHH) Bar of Gold Clam shell appearance Hexagonal w/ blunt ends and stain darkly Solubility Washington monument shape Dark-hued crystal of condensed Hgb distorts the RBC membrane Solubility Nucleated RBC (immature) that contains nonheme iron particles Page | 188
Siderocyte Pappenheimer bodies
Malaria
Babesia Agglutination Rouleaux
Drop of NSS Hemoglobinopathies HbS HbC HbE Sickle cell anemia Sickle cell trait Thalassemia α-thalassemia β-thalassemia
Requires Prussian blue stain ♫ Sideroblastic anemia (Iron overload) ♫ MDS Non-nucleated RBC (mature) containing iron granules (hemosiderin) ♫ Sideroblastic anemia ♫ MDS Iron granules visible w/ Wright’s stain Resembles basophilic stippling = PB: Periphery = BS: Homogeneous Unused iron deposits ♫ Sideroblastic anemia ♫ MDS MOT: Anopheles mosquito bite Maturation stages: Rings > Trophozoite > Schizonts > Gametocytes P. vivax = Worldwide P. falciparum = Philippines Resistant to Malaria: = Fy(a-b-): African = Sickle cell anemia = G6PD deficiency MOT: Tick bite “Maltese cross” Resemble P. falciparum rings Cold agglutinin disease (anti-I) Primary atypical pneumonia (anti-I) RBCs in stack of coins arrangement plasma globulin ♫ Multiple myeloma: proliferation of Ig-producing plasma cells (ESR) ♫ Macroglobulinemia Disperses rouleaux formation True agglutination remains intact Hemoglobinopathies Qualitative defect in Hgb Ex. Substitution α2β26 Glu Val α2β26 Glu Lys α2β226 Glu Lys 80/90-100% HbS HbF & HbA2 55-60% HbA1 |40-45% HbS Thalassemia Quantitative defect in Hgb Alpha-globin chain is or absent a. Adult = β4 = HbH (deletion of 3/4 alpha genes) b. Neonate = γ4 = Hb Bart’s (deletion of 4/4 alpha genes) Beta-globin chain is or absent HbF and HbA2 Page | 189
Cooley’s anemia Cooley’s trait Cellulose acetate Hgb electrophoresis
Citrate agar Hgb electrophoresis
Screening test for HbS
HbA2 HbF
Tests for unstable Hgb (HbH) Morphology Characteristics Macrocytes (>9 μm) Microcytes (<9 μm) Hypochromia Poikilocytosis Polychromatophilia Rouleaux Pelger-Huet
Thalassemia major Homozygous β-thalassemia Thalassemia minor Heterozygous β-thalassemia Alkaline pH: 8.6 Migration: (Cathode > Anode) C > S > F > A1 > Barts > I > H E D O G A2 Lepore Normal: HbA1 is the fastest (most anodal) Abnormal: HbH is the fastest (most anodal) Acid pH: 6.0-6.3 Migration: (Cathode > Anode) F > A |Origin| O > S > C E D G 1. Sodium metabisulfite = (+) Sickling of cells 2. Solubility test = Sodium thiosulfite = (+) Turbidity in β-thalassemia Quantitation: Anion exchange microchromatography Alkali resistant (+) HiCN Tests: 1. Alkali denaturation test = HbF resists alkali denaturation a. Betke (NaOH) b. Singer (KOH) 2. Acid elution test = HbF resists acid-elution = Cells w/ HbF = deep pink color = Cells w/ N-HbF = ghost cells 1. Heat precipitation test: Δ50’C for 2 hrs 2. Isopropanol precipitation test: 17% solution Sample Criteria for Erythrocyte Morphology Evaluation w/in Normal 1+ 2+ 3+ Limits (OIO) (per OIO) (per OIO) (per OIO) 0-5 5-10 10-20 20-50 0-5 5-10 10-20 20-50 0-2 3-10 10-50 50-75 0-2 3-10 10-20 20-50 -1-5 6-10 >10 Numerous -Agg. of 3-4 RBCs Agg. of 5-10 RBCs aggregates
4+ (per OIO) >50 >50 >75 >50 ---
Nuclear Abnormalities Hyposegmentation (neutrophil) Bilobed nucleus: Dumb-bell shaped/spectacle/peanut-shaped/”Pince-nez” Page | 190
Hypersegmentation
Alder-Reilly granules
Toxic granules Toxic vacuoles Auer rods Faggot cells Chediak-Higashi granules
May-Hegglin inclusion
Dohle bodies Dohle-Amato bodies
IT: Infections, Toxic states
Job’s syndrome Lazy leukocyte syndrome Chronic Granulomatous Disease (CGD)
Resembles Stab cell (To differentiate: PH cell has more clumped chromatin) ♫ Pelger-Huet anomaly = Autosomal Dominant ♫ Pseudo-Pelger-Huet = Acquired in myeloproliferative disorders ≥ 6 lobes (neutrophil) Abnormal DNA synthesis ♫ Undritz anomaly = hereditary hypersegmentation ♫ Megaloblastic anemia Cytoplasmic Abnormalities Large purple-black coarse cytoplasmic granules Accumulation of degraded mucopolysaccharides (all leukocytes) ♫ Alder-Reilly anomaly = Autosomal Recessive ♫ Mucopolysaccharidoses: Hurler, Hunter, Sanfilippo syndrome Resemble toxic granules (IT) Large purple to black granules resembling ALR granules ♫ Infections ♫ Toxic states Infections Toxic states Pink or red rod shaped structures Fused primary granules (peroxidase positive) Myeloid and monocytic series only w/ mass of Auer rods M3 (APL) = associated w/ DIC Giant red, blue to grayish round inclusions (large lysosomal granules) Seen in lymphocyte, neutrophil and monocyte Lysosomal defects Platelets lack dense granules ♫ Chediak-Higashi syndrome = Autosomal Recessive (Albinism) Pale blue inclusions derived from RNA ♫ May-Hegglin anomaly = Autosomal Recessive = Giant platelets = Thrombocytopenia Resemble Dohle bodies (IT) Single or multiple blue inclusions Aggregates of free ribosomes of rough ER Resembles ♫ Infections ♫ Toxic states Dohle bodies Toxic granules Toxic vacuoles Abnormalities in Function Normal random activity Abnormal chemotactic activity Abnormal random and chemotactic activity Inability of phagocytes to kill ingested microorganisms Impaired NADPH oxidase Impaired oxidative metabolism/respiratory burst Page | 191
LE cell
Tart cell
Test: NBT dye test Cells Exhibiting Phagocytosis Neutrophil w/ large purple homogeneous round inclusion Believe to be a neutrophil that ingested another neutrophil Buffy coat Smooth and evenly stained ♫ SLE Monocyte w/ ingested lymphocyte Rough and unevenly stained
Abnormalities Involving Lymphocytes a. Type I = Turk’s irritation cell = Plasmacytoid lymphocyte w/ large block of chromatin b. Type II = Infectious mononucleosis: caused by EBV (target: B cells [CD21]) = Atypical lymphocyte in IM: T cells reacting to B cells infected w/ EBV c. Type III = Vacuolated = Swiss cheese/moth eaten appearance Basket cell Lymphocyte w/ thumbprint appearance Smudge cell ♫ Due to pressure in smear preparation Automated cell count ♫ Remedy: Add bovine albumin ♫ CLL Hairy cells B cells w/ hair-like projection ♫ Hairy cell leukemia = TRAP (+) Sezary cells w/ cerebriform nucleus (“brain-like”) ♫ Sezary syndrome ♫ Mycosis fungoides Abnormalities Involving Monocytes/Macrophages/Histiocytes (Lipidoses/Lipid Storage Diseases) Gaucher’s disease Accumulation of glucocerebroside (-) glucocerebrosidase/β-glucosidase Wrinkled/crumpled cytoplasm (Chicken scratch) Niemann-Pick disease Accumulation of sphingomyelin (-) sphingomyelinase Foamy cytoplasm Foam cells w/ sphingomyelin Tay Sach’s disease Accumulation of glycolipid and ganglioside (-) Hexosaminidase A Vacuolated cytoplasm Sandhoff’s disease Accumulation of glycolipid and ganglioside (-) Hexosaminidase A & B Vacuolated cytoplasm Sea blue histiocytosis Unknown enzyme deficiency Blue-green cytoplasm Abnormalities Associated w/ Plasma Cells Flame cell Plasma cell w/ red to pink cytoplasm Page | 192 Reactive lymphocyte Atypical lymphocyte Stimulated lymphocyte Variant lymphocyte Downey cell
Grape cell Mott cell Morula cell Berry cell Russell bodies Dutcher’s bodies Giant platelet Small/micromegakaryocyte Large megakaryocyte Mononuclear megakaryocyte Vacuolated megakaryocyte Leukemia
Acute leukemia Subacute leukemia Chronic leukemia Leukemic leukemia Subleukemic leukemia Aleukemic leukemia French-American-British (FAB) Classification of Acute Leukemias Acute leukemia Acute leukemia in children Tests to differentiate ALL from ANLL
♫ Multiple myeloma of IgA origin Plasma cell w/ vacuoles Accumulation of Russell bodies ♫ Multiple myeloma Individual globules of immunoglobulin Intranuclear protein inclusions Platelet Abnormalities (Morphologic) ♫ Bernard-Soulier syndrome ♫ May-Hegglin anomaly ♫ Myelodysplastic syndromes
Leukemia Abnormal, uncontrolled proliferation and accumulation of one or more of the hematopoietic cells Symptoms: Fever, weight loss, sweating; hepatosplenomegaly, enlarged lymph nodes (chronic leukemia) BMR Days to 6 months Predominantly immature cells (blasts and “pro” stages) 2 to 6 months Variable Minimum of 1 or 2 years Predominantly mature cells WBC >15,000/μL WBC <15,000/μL (+) Abnormal and immature cells in PB WBC <15,000/μL (-) Abnormal and immature cells in PB Divides acute leukemias into lymphoblastic and monoblastic = Subdivided according to cellular morphology, cytochemical staining results, cytogenetic studies and T & B lymphocytes marker results Normocytic, normochromic RBCs FAB = ≥30% blasts Henry: WHO (Now the standard for diagnosis) = ≥20% 80% ALL 20% ANLL 1. MPO: Myeloperoxidase = (+) AML = (-) ALL 2. SBB: Sudan Black B = (+) AML = (-) ALL 3. TdT: Terminal Deoxyribonucleotidyltransferase = Marker for immature lymphocyte Page | 193
L1 L2 L3
M1
M2
M3
M4 M5a M5b M6
M7
MPD 1. Chronic Myelogenous Leukemia (CML)
= (+) ALL = (-) ANLL Acute Lymphoblastic Leukemias (ALL) Lymphoblasts are small and homogeneous (vary little in size) Childhood ALL Lymphoblasts are large and heterogeneous (vary in size) Adult ALL Burkitt-type Rare Lymphoblasts are large but homogeneous, and vacuolated Acute Nonlymphocytic Leukemias (ANLL) Acute myeloblastic leukemia w/o maturation (AML w/o mat) BM: >30% blasts <10% granulocytic cells Acute myeloblastic leukemia w/ maturation (AML w/ mat) BM: >30% blasts >10% granulocytic cells Acute promyelocytic leukemia (APL) >30% blasts >10% granulocytic cells >30% or >50% promyelocytes (+) Faggot cells = Associated w/ DIC Acute myelomonocytic leukemia (AMML) Naegeli’s leukemia 20% to <80% monocytic cells Acute monoblastic leukemia w/o maturation Schilling’s leukemia >80% monocytic cells (>80% monoblasts) Acute monoblastic leukemia w/ maturation >80% monocytic cells (<80% monoblasts) Erythroleukemia Erythremic myelosis Di Guglielmo’s syndrome >30% blasts >50% erythrocytic precursors Acute megakaryocytic leukemia >30% blasts >30% megakaryocytic cells Chronic Myeloproliferative Disorders Proliferation of abnormal pluripotential stem cell Stem cell differentiates into the granulocytic (myeloid stem cell), megakaryocytic and erythroid cell lines (+) Philadelphia chromosome: t(9+;22-) - both long arms If (-) Ph’ chromosome = poor prognosis Similar to leukomoid reaction, to differentiate: a. Chromosome studies b. LAP = ( in Leukomoid reaction, in CML)
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2. Myelofibrosis w/ myeloid metaplasia (MMM)
Fibrosis and granulocytic hyperplasia of BM, w/ granulocytic and megakaryocytic proliferation in the liver and spleen (extramedullary) (+) Dacryocytes LAP BM aspirate = impossible (dry tap) BM biopsy = appropriate 3. Essential Thrombocytosis: 1,000 x 109/L Thrombocythemia (ET) Functionally abnormal platelets 4. Polycythemia Vera (PV) BM: Panmyelosis PB: Pancytosis/Pancythemia RBCs, WBCs, Plts LAP (Other polycythemia: N-LAP) Polycythemia 1’ Absolute polycythemia Other names: Polycythemia Vera, Polycythemia Rubravera, Vaquez Osler disease, Panmyelosis RBC mass ( Hct) RBCs, WBCs, Platelets Erythropoietin (EPO) 2’ Absolute polycythemia In response to hypoxia w/ appropriate production In patients w/ pulmonary/cardiac disease of EPO RBCs, WBCs, Platelets EPO 2’ Absolute polycythemia In patients w/ tumors of kidney, liver, brain, adrenal and pituitary gland w/ inappropriate RBCs, N-WBCs, N-Platelets production of EPO EPO Relative polycythemia Spurious/Gaisboö ck polycythemia Associated w/ stress and anxiety N-RBC mass Hct because of decreased plasma volume RBC mass Differentiate absolute from relative polycythemia RBC mass = Absolute polycythemia N-RBC mass = Relative polycythemia Myelodysplastic Syndrome/Dysmyelopoietic Syndrome MDS Clonal abnormalities in hematopoietic cells “Pre-leukemia”: can progress to ANLL if not treated <30% blast Differentiation % Blasts in PB % Blasts in BM Comments Refractory anemia Mildest type <1% <5% (RA) RA w/ ringed sideroblast <5% RS: Ringed sideroblast <1% (RARS) >15% RS RA w/ excess blast <5% 5-20% (RAEB) RAEB in transformation >5% 20-30% (RAEBt) Chronic Myelomonocytic Persistent monocytosis <5% 5-20% Leukemia (CMML) Lymphoproliferative disorders Page | 195
LPD 1. T/B cell leukemia 2. Lymphoma
3. Hairy cell leukemia
4. Mycosis fungoides and Sezary syndrome Myeloperoxidase (MPO)
Sudan Black B (SBB)
Terminal deoxyribonucleotidyl Transferase (TdT) Periodic Acid-Schiff (PAS)
Naphthol AS-D Chloroacetate esterase
α-Naphthyl Acetate esterase
Proliferation of cells derived from lymphoid stem cell T/B cells -Malignancy involving lymphoid tissue a. Non-Hodgkin’s lymphoma = proliferation of neoplastic lymphocytes = Rappaport classification b. Hodgkin’s lymphoma = proliferation of cells reacting to neoplasm = (+) Reed-Sternberg cell: large cell w/ large nucleoli (Owl’s eye) ♫ Diagnosis: Lymph node biopsy = Rye classification: based on histologic appearance of lymph node biopsy = Ann-Arbor: staging based on the extent of tissue involved Leukemic reticuloendotheliosis Originally B cells w/ hairlike projections (+) TRAP: Tartrate-resistant acid phosphatase Neoplastic T cells Sezary cells: w/ cerebriform nucleus (Brainlike) Special Stains Marker for primary granules and Auer rods (+) AML (-) ALL Stain should only be done on fresh specimens Marker for phospholipids and lipids (+) AML (-) ALL Can be done on stored specimens Parallels MPO for interpretation DNA polymerase immunoperoxidase Marker for immature lymphocytes (+) ALL (-) AML Marker for glycogen, glycoproteins, mucoproteins and HMW CHO a. Normal: all blood cells are PAS (+) except erythroblast b. Abnormal: erythroblasts are PAS (+) = M6 (+) L1 and L2 = blocklike positivity (-) L3 (+) M6 Specific esterase Marker for mature and immature neutrophils and mast cells (+) AML (-) AMoL [M5] (-) ALL Lasts for months Nonspecific esterase Marker for monocytes, megakaryocytes and plasma cells (+) AMoL [M5] = inhibited by fluoride (+) AMegL [M7] (-) AML Page | 196
α-Naphthyl Butyrate esterase Acid phosphatase
Tartrate resistant acid phosphatase (TRAP) Leukocyte alkaline phosphatase (LAP)/ Neutrophil ALP (NAP)
Toluidine blue
Cooper & Cruickshank Nitroblue tetrazolium test (Original)
Modified NBT
Feulgen reaction Prussian blue stain
Nonspecific esterase Identifies monocytes, promonocytes and monoblasts (+) AMoL [M5] = inhibited by fluoride (-) AML Present in all hematopoietic cells and found in lysosomes (+) Hairy cell leukemia = TRAP (+) (+) T cell leukemia (-) Non-T cell leukemia Cannot be stored Excellent marker for hairy cell leukemia Neutrophil is the only leukocyte that contains this activity Found in 3’ granules (Metamyelocytes) Leukomoid reaction, MMM, PV, 3rd trimester of pregnancy CML, CGL, PNH Should be done on fresh specimen ♫ Kaplow count: = Count 100 neutrophils = Grade 0 to 4+ NV = 30-185 LAP Score: 0 = No stain 1+ = Faint stain 2+ = Pale stain 3+ = Strong stain 4+ = Deep (Very intense) stain Binds w/ acid mucopolysaccharides in blood cells Strongly metachromatic Useful for recognition of mast cells and tissue basophils (metachromatic granules) CGL: Chronic granulocytic leukemia Stain for basophils Reagent: Toluidine blue Test for CGD NBT: Colorless to pale yellow ---(Toxic O2 molecules)---> (+) Blue formazan Test: ♫ Heparinized blood Centrifuge Buffy coat (WBC) + NBT (+) Formazan NV = ≤10% formazan (+) CGD = 0 to negligible Infection = 70% w/ stimulating agent Test: ♫ Buffy coat (WBC) + Bacterial suspension ---(NBT)---> (+) Formazan NV = 80-100% formazan CGD = <50% formazan Specific reaction for DNA Howell-Jolly bodies = Feulgen (+) Stain for hemosiderin (+) Sideroblast Page | 197
Supravital stain (RHH)
BCB
(+) Siderocytes RHH = NCB Reticulum = New methylene blue Heinz bodies = Crystal violet Hemoglobin H = Brilliant cresyl blue Stain for automated cell counter
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Primary hemostasis Platelets
Secondary hemostasis Arteries Veins Capillaries Maturation Stage Megakaryoblast Promegakaryocytes Megakaryocytes Metamegakaryocytes Platelet structure
Platelet Adhesion
Bernard-Soulier syndrome
HEMOSTASIS Involves blood vessels and platelets Formation of platelet plug Test: Bleeding time Functions: Adhesion Activation Release Aggregation Involves the coagulation factors Formation of stable fibrin clot Test: Clotting time Carry blood from the heart to the capillaries Primary Hemostasis Return blood from the capillaries to the heart Injured vessel: vasoconstriction = initiated by serotonin and thromboxane A2 derived from platelets and endothelial cells Cytoplasmic Cytoplasmic Nuclear Features Thrombocytes Granules Tags Visible (-) (+) Single nucleus (-) Fine chromatin (+) Nucleoli Few (+) 2 nucleus (-) Numerous Usually (-) 2 or more nuclei (-) Aggregated (-) 4 or more nuclei (+) 60% proteins 30% lipids 8% carbohydrate Various minerals, water, nucleotides 1. Peripheral zone = responsible for adhesion and aggregation a. Glycocalyx = outer surface b. Plasma membrane = consists of 30 or more glycoprotein c. Submembranous area 2. Sol-gel zone = platelet shape & contractile elements a. Microfilaments: actin & myosin (actomyosin/thrombostenin) = responsible for clot retraction b. Microtubules = consists of tubulin: maintain the platelet shape 3. Organelle zone = alpha & dense granules = mitochondria, lysosomal granules 4. Membranous system a. Dense tubular system = site of arachidonic acid metabolism b. Open canalicular system (surface connecting system) = release of granules Platelet adherence to exposed subendothelial surface (collagen) Occurs in the presence of von Willebrand factor In vivo: collagen In vitro: glass (-) gpIb = receptor for vWF Page | 199
(Giant platelet syndrome) Von Willebrand disease Platelet Activation
(-) vWF = for platelet adhesion Morphologic and functional changes in platelets Agonists: substance that initiate activation Arachidonic acid ---(Cyclooxygenase)---> Thromboxane A2 TxA2 Vasoconstrictor Stimulate platelet secretion Aspirin Inhibits COX bleeding time Platelet Secretion Release of granules a. Alpha granules = Platelet factor = Platelet derived growth factor = Fibrinogen = Factor V = vWF = β-thromboglobulin = Thrombospondin = Fibronectin = Albumin b. Dense granules (CAPAS) = Calcium = ADP = Pyrophosphate = ATP = Serotonin Release disorders Storage pool defects: a. Alpha-granule deficiency ♫ Gray platelet syndrome ♫ Quebec platelet disorder = (-) Factor V binding protein (multimerin) b. Dense granule deficiencies ♫ Hermansky-Pudlak ♫ Chediak-Higashi ♫ Wiskott-Aldrich syndrome (α & dense granule deficiency) Important Substances Secreted by Platelets & Their Role in Hemostasis Promote coagulation HMWK (α) Fibrinogen (α) Factor V (α) Factor VIII:vWF (α) Promote aggregation ADP (d) Calcium (d) Platelet factor 4 (α) Thrombospondin (α) Promote vasoconstriction Serotonin (d) Thromboxane A2 precursors (mb.PL) Promote vascular repair Platelet-derived growth factor (α) = promotes smooth muscle growth β-thromboglobulin (α) = chemotactic for fibroblasts Other systems affected Plasminogen (α) α2-antiplasmin (α) Page | 200
Platelet aggregation Glanzmann’s thrombasthenia Petechiae Purpura Ecchymosis Epistaxis Hemarthrosis Hematemesis Hematoma Hematuria Hemoglobinuria Melena Menorrhagia Hereditary hemorrhagic telangiectasia (Oslwer-Weber-Rendu disease) Congenital hemangiomata (Kasabach-Merritt syndrome) Ehler-Danlos syndrome Marfan’s syndrome Pseudoxanthoma elasticum Senile purpura Scurvy Henoch-Schonlein purpura Thrombocytopenia
Thrombocytosis
Platelet adhesion
C1 esterase inhibitor (α) Platelet attachment to each other Requires fibrinogen and Ca2+ (-) gpIIb/IIIa complex: receptor for fibrinogen Pinpoint hemorrhagic spots Hemorrhage of blood into small areas of skin Blood escapes into large areas of skin Nosebleed Leakage of blood into joint cavities Vomiting of blood Swelling or tumor in the tissues or a body cavity that contains clotted blood RBC in urine Hgb in urine Stool containing dark red or black blood Excessive menstrual bleeding Vascular Disorders Most common inherited vascular disorder Blood vessels are thin & lack smooth muscle Tumor composed of blood vessels vascular fragility Elastic fibers are calcified & structurally abnormal Degradation of collagen & elastin (-) Vitamin C = for collagen synthesis Defective synthesis of collagen Immunologic damage to endothelial cells Quantitative Platelet Disorders Platelet production of BM = aplastic anemia Survival time = platelet destruction (DIC, ITP) Platelet sequestration by the spleen = splenomegaly Dilution of platelet count = Thrombocytopenia α # of units transfused Units transfused = Thrombocytopenia Multiple transfusion: stored blood contains nonviable platelets Reactive = moderate increase, asymptomatic (after hemorrhage, splenectomy) Autonomous = marked increase, associated w/ thrombotic/hemorrhagic complications (Ex. ET: platelet function is abnormal) Qualitative Platelet Disorders 1. vWD = (-) vWF ♫ Platelet aggregation test: = Normal: Epinephrine, Collagen, ADP = Abnormal: Ristocetin 2. BSS = (-) gpIb (+) Giant platelets Page | 201
Acquired
Platelet count
Unopette
Platelet estimate (Wedge smear) WBC estimate (HPF) Platelet Estimate of 0-49,000/μL
♫ Platelet aggregation test: = Normal: Ristocetin = Abnormal: Epinephrine, Collagen, ADP 3. Storage pool defects = Gray platelet (α), HP, WAS, CHS (d) Uremia: toxic metabolites Paraproteinemias: coating of platelet membrane w/ abnormal protein AML: abnormal megakaryocytes MPD Drugs: Aspirin = inhibits COX
Laboratory Tests for Primary Hemostasis 1. Direct (Neubauer counting chamber) Plt. count/mm3 = # plt x AF x DCF x DF = # plt x 1 x 10 x 200 = # plt x 2,000 (RBC pipette = 1:200 dilution) ♫ 1 platelet uncounted = -2,000 plt/mm3 a. Reese-Ecker - Sodium citrate - Formaldehyde - Brilliant cresyl blue b. Guy and Leake - Sodium oxalate - Formaldehyde - Crystal violet c. Brecker-Cronkite - 1% ammonium oxalate (phase contrast microscopy) -----------------------------------------------------------------------------------------------------d. Unopette 2. Indirect (smear) Platelet count = (# of plts ÷ 1000 RBC) x RBC count a. Dameshek b. Fonio’s c. Olef’s Diluent: 1% Ammonium oxalate Stand moist chamber for 15-20mins to allow platelets to settle 1.98mL diluent 0.02mL blood TV = 2mL Dilution = 1:100 Plt. count/mm3 = # plt x ACF x DCF x DF = # plt x 1 x 10 x 100 = # plt x 1,000 ♫ 1 platelet uncounted = -1,000 plt/mm3 8-20 platelets/OIO Factor: 20,000 Factor: 2,000 Platelet Estimates (Smear) Report Platelet Estimate as Markedly decreased Page | 202
50,000-100,000/μL 100,000-150,000/μL 150,000-199,000/μL 200,000-400,000/μL 401,000-599,000/μL 600,000-799,000/μL >800,000μL N-Plt count, BT Plt count, N-BT Plt count, BT Platelet aggregation test
Platelet adhesiveness (Salzmann)
Clot retraction time
Capillary resistance test Touriquet test Rumpel-Leedes test
Bleeding time
Coagulation factors
Moderately decreased Slightly decreased Low normal Normal Slightly increased Moderately increased Markedly increased Qualitative platelet abnormality Primary vascular abnormality vWD Autoimmune thrombocytopenia Simultaneous quantitative and qualitative platelet deficiency Aggregating agents: a. Epinephrine b. Collagen c. ADP d. Ristocetin Sample: Citrated blood Centrifuge at 60-100g for 10mins PRP + Agonist O.D. monitored (Aggregometer) Determination of in vitro platelet adhesion Plt ct. 1 = routine collection method Plt ct. 2 = collected through glass bead collecting system % PA = [(PC1-PC2) ÷ PC1] x 100 NV = 26-60% in vWD Proportional to platelet count Clot retraction: function of thrombosthenin a. Castor oil/Hirschboeck (+) Dimpling/droplet like serum on the surface of blood drop NV = 15-45mins b. Stefanini c. MacFarlane CRT = (vol. serum ÷ TV) x 100 NV = 44-67% Measures capillaries to resist pressure Correlates w/ the degree of thrombocytopenia 100 mmHg for 5 mins After 15-30mins, count petechiae (or halfway bet. systolic & diastolic pressure for 5 mins) +1 = 0-10 petechiae (few) +2 = 10-20 petechiae (many) +3 = 20-50 petechiae (multiple) +4 = >50 petechiae (confluent) NV = 0 to occasional Not repeated on the same arm for 7 days In vivo measure of primary hemostasis a. Duke method = fingertip/earlobe b. Ivy method = uses blood pressure cuff (40mmHg) puncture forearm Secondary Hemostasis All are produced in the liver except Factor VIII Page | 203
Numeral I II III IV V VII VIII:C IX X XI XII XIII
-
Stage I: Generation of Thromboplastin Stage II: Conversion of prothrombin to thrombin Stage III: Conversion of fibrinogen to fibrin clot Fibrinogen Calcium
In liver disease, all coagulation factors are except factor VIII complex Factor VIII complex: a. VIII: Coagulant (VIII:C) b. vWF: produced by megakaryocytes and endothelial cells Activated at cold temp. = VII & XI Coagulation Factors Preferred Name Synonyms Fibrinogen Prothrombin Prethrombin Tissue factor Tissue thromboplastin Calcium Proaccelerin Labile factor Accelerator globulin (aCg) Proconvertin Stable factor Serum prothrombin conversion accelerator (SPCA) Antihemophilic factor Antihemophilic factor globulin (AHG) Antihemophilic factor A Platelet cofactor 1 Plasma thromboplastin component Christmas factor Antihemophilic factor B Platelet cofactor 2 Stuart-Prower factor Stuart factor Prower factor Autoprothrombin III Plasma thromboplastin component Antihemophilic factor C Hageman factor Glass factor Contact factor Fibrin stabilizing factor Laki-Lorand factor Fibrinase Plasma transglutaminase Fibrinoligase Prekallikrein Fletcher factor High-molecular weight kininogen Fitzgerald factor Contact activation cofactor Williams factor Flaujeac factor Intrinsic: XIIXIIX—VIII (Cofactor) Extrinsic: IIIVII Common: X—V (Cofactor) ----> Common thromboplastin/Prothrombinase (Va-Xa-Ca2+-PL) II ---(Prothrombinase)---> Thrombin I ---(Thrombin)---> Fibrin clot XIII---(Thrombin)---> XIIIa Fibrin clot ---(XIIIa)---> Stable fibrin clot Most concentrated Involved in all stages of coagulation except contact phase Page | 204
Contact group
XII, XI, PK, HMWK Ca2+ independent Vit. K independent Involved in the contact phase XII ---(Collagen)---> XIIa (small amount) PK ---(XIIa)--------> Kallikrein XII ---(Kallikrein+HMWK)---> XIIa (large amount) XI ---(XIIa)---------> XIa Fibrinogen group I, V, VIII, XIII Ca2+ dependent Vit. K independent Completely consumed during coagulation (+) in plasma (-) in serum Prothrombin group II, VII, IX, X Ca2+ and Vit. K dependent First: VII IX X II: Last Adsorbable factors: removed by adsorbing agents [BaSO4, Al(OH)3] (+) in plasma (-) in serum Diseases BT PT APTT Stypven TT Duckert’s Disease of 1’ hemostasis N N N N N Fibrinogen deficiency N* N Prothrombin deficiency N N N Parahemophilia N N N Factor VII deficiency N N N N N Hemophilia A N N N N N von Willebrand disease N N N N Hemophilia B N N N N N Factor X deficiency N N N Hemophilia C N N N N N Factor XII deficiency N N N N N Factor XIII deficiency N N N N N Abn DIC Abn *BT may be prolonged in afibrinogenemia Adsorbed Fresh Plasma Aged Plasma Fresh Serum Aged Serum Plasma I + + + II + + + (<20%) V + + VII + + + + VIII + + IX + + + + X + + + + XI + + + + + XII + + + + + XIII + + + Prothrombin: Page | 205
80% is consumed during coagulation <20% residual prothrombin Disorders of Coagulation Causing Clotting Factor Deficiencies Inherited Coagulopathies Acquired Coagulopathy Factor Inheritance Pattern Coagulopathy I Autosomal recessive Afibrinogenemia Liver disease Autosomal dominant Dysfibrinogenemia DIC Fibrinolysis II Autosomal recessive Prothrombin deficiency Liver disease Vit. K deficiency Anticoagulant therapy V Autosomal recessive Owren’s disease Liver disease Labile factor deficiency DIC Parahemophilia Fibrinolysis VII Autosomal recessive Factor VII deficiency Liver disease Vit. K deficiency Anticoagulant therapy VIII X-linked recessive Hemophilia A DIC Autosomal dominant vWD Fibrinolysis IX Autosomal recessive Hemophilia B Liver disease Christmas disease Vit. K deficiency Anticoagulant therapy X Autosomal recessive Factor X deficiency Liver disease Vit. K deficiency Anticoagulant therapy XI Autosomal recessive Hemophilia C Unclear Rosenthal syndrome =Common in Jewish descent/ Ashkenazi Jews XII Autosomal recessive Factor XII deficiency Unclear =No bleeding tendency XIII Autosomal recessive Factor XIII deficiency Liver disease DIC Fibrinolysis PK Autosomal recessive Fletcher trait Unclear HMWK Autosomal recessive Fitzgerald trait Unclear Factor VIII deficiency Common inherited coagulation factor deficiency a. Hemophilia A/Classic hemophilia/Royal disease = Queen Victoria’s son b. vWD = most frequently inherited coagulation disorder Major sites of coagulation Endothelium inhibition Platelet Protein C Degrades factor Va and VIIIa Protein S Vit. K dependent glycoprotein Antithrombin Major inhibitor of thrombin Tests for Secondary Hemostasis Clotting time Measure the period required for free form of blood to clot after it has been removed from the body a. Capillary blood method Page | 206
Prothrombin time
Activated partial thromboplastin time
Stypven time/Russel viper venom time
Thrombin time
Reptilase time
Duckert’s test 5M urea solubility test
= Drop or slide = Capillary tube/Dale and Laidlaw = Blue capillary tube NV = 2-4mins b. Whole blood/Lee and White NV = 7-15mins Detect coagulation factor deficiencies involving extrinsic and common pathway Citrated blood Centrifuge at 2000g for 10mins PPP PPP + Thromboplastin-CaCl2 rgt (+) Clot Begin timing for clot formation on the addition of CaCl 2 rgt NV = 10-12 secs Detect coagulation factor deficiencies involving intrinsic and common pathway PPP + APTT rgt: 1. Activators: a. Micronized silica b. Ellagic acid c. Celite d. Kaolin 2. Phospholipids: substitute for platelets 3. CaCl2 Begin timing for clot formation on the addition of CaCl 2 rgt NV = 25-35 secs East Indian viper venom Vipera russelli: directly activates factor X Detect coagulation factor deficiencies involving common pathway PPP + Stypven rgt: platelin-CaCl2 rgt NV = 6-10 secs Prolonged in: a. Fibrinogen deficiency b. Presence of FDP or FSP c. Presence of thrombolytic agent (Ex. streptokinase) d. Presence of heparin PPP + Thrombin-CaCl2 rgt NV = 10-14 secs Enzyme found in the venom of Bothrops atrox snake Prolonged in: a. Fibrinogen deficiency b. Presence of FDP or FSP c. Presence of thrombolytic agent Not affected by heparin PPP + Atroxin Begin timing for clot formation upon the addition of atroxin NV = 10-15 secs For factor XIII deficiency Rgt: 5M Urea Substitutes for urea: - 1% monochloroacetic acid - 2% acetic acid Normal = Clot is insoluble to urea (24 hrs) Page | 207
Circulating anticoagulants Lupus inhibitor Tilt tube method Fibrometer Photo-optical detection of clot formation
Fibrinolysis Plasminogen activators
Inhibitors of fibrinolysis
Degradation of cross-linked fibrin by plasmin
Degradation of noncrosslinked fibrin and fibrinogen by plasmin
F. XIII def. = Clot is soluble to urea (24 hrs) Prolonged APTT and PT not corrected Against the phospholipid portion of prothrombinase complex (Va-Xa-Ca 2+-PL) Instrumentation for Tests of Hemostasis Visual detection of fibrin clot formation Manual technique End point = clot formation Electromechanical detection of fibrin clot formation Fibrin strand formation is detected using a wire loop or hook w/c has been incorporated into a semi-automated mechanical instrument Detection of fibrin clot formation depends on in light scattering associated w/ conversion of fibrinogen fibrin ♫ Semi-automated instruments: - Electra 750 and 750A - Fibrintimer series - FP 910 coagulation analyzer ♫ Automated instruments: - Ortho Koagulab 16S and 40A - Coag-A-Mate X2 and XC - MLA Electra 700 and 800 Digestion of fibrin clot Occurs when plasminogen plasmin 1. Intrinsic activators - XIIa - Kallikrein - HMWK 2. Tissue type - Urokinase-like PA 3. Therapeutic activators = treatment for thromboemboli - Streptokinase - Urokinase - Tissue-like PA α2-antiplasmin = Major inhibitor α2-macroglobulin Thrombospondin PA inhibitor 1 and 2 Crosslinked fibrin (urea insoluble) ↓ (Plasmin) ↓ Complex DD/E | Complex YD/DY | Complex YY/DXD (D-dimer) Fibrinogen or Noncrosslinked fibrin (urea soluble) ↓ (Plasmin) ↓ Fragment X ↓ Page | 208
Primary fibrinolysis
Secondary fibrinolysis
Whole blood clot lysis time (WBCLT) Euglobulin lysis time
Protamine sulfate test Ethanol gelation test Latex D-dimer assay Test WBCLT Euglobulin clot lysis Ethanol gelation Protamine sulfate D-dimer Heparin
(Plasmin) ↓ Fragment Y | Fragment D ↓ (Plasmin) ↓ Fragment D | Fragment D
No fibrin monomer No fibrin polymer No D-dimer Plasminogen activators from damaged/malignant cells Converts plasminogen plasmin in the absence of fibrin formation ♫ Prostatic carcinoma DIC = uncontrolled, inappropriate formation of fibrin w/in the blood vessels w/ fibrin monomer w/ fibrin polymer w/ D-dimer = most specific for DIC ♫ Infection: N. meningitidis ♫ Neoplasm ♫ Snake bite ♫ HTR Laboratory Evaluation of Fibrinolysis Clot should remain intact for approximately 48 hrs at 37’C Clot lysis prior to 48 hrs indicates excessive systemic fibrinolysis Euglobulins: proteins that ppt. when plasma is diluted w/ H2O & acidified - Plasminogen - Plasmin - Fibrinogen - Plasminogen activators Plasma + Acetic acid Ppt. euglobulin Euglobulin + thrombin Clot euglobulin Euglobulin Dissolved in buffer Normal = No clot lysis (2 hrs) Fibrinolysis = Clot lysis <2 hrs Detects the presence of fibrin monomers (2’-DIC) in the plasma Plasma + Protamine sulfate + ETOH (+) gel-like clot (paracoagulation) Detects the presence of fibrin monomers (2’-DIC) in the plasma Plasma + NaOH (pH) + ETOH (+) pptn/gel Most specific test for DIC Primary Fibrinolysis Secondary Fibrinolysis < 48 hrs < 48 hrs < 2 hrs < 2 hrs + + + Injected Action: inhibits thrombin Monitoring: APTT = sensitive, method of choice (CAP) Neutralize w/ protamine sulfate Page | 209
Warfarin Coumarin Coumadin INR
ISI
Oral WARF: Wisconsin Alumni Research Foundation Action: Vit. K antagonist, inhibits II, VII, IX, X Monitoring: PT (reported in INR) Neutralize w/ Vitamin K, FFP International normalized ratio INR = (Patient PT ÷ Normal PT)ISI ♫ INR = 2-3 - Prevents MI, embolism & thrombosis ♫ INR = 2.5-3.5 - For patients w/ mechanical heart valves International Sensitivity Index (PT rgt) PT rgt is calibrated w/ Manchester rgt = from human brain thromboplastin
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Brightfield microscopy Oil immersion microscopy Phase-contrast microscopy Fluorescence microscopy Electron microscopy Basic component of Standard light microscope
Total magnification
Hemoglobin
Acid hematin Alkali hematin Gasometric (Van Slyke) Chemical (Kennedy’s, Wong’s) SG method Cyanmethemoglobin/ Hemiglobincyanide method
HEMATOLOGY PROCEDURES Examine blood films Erythrocyte morphology & leukocyte differential For manual platelet counts ANA and T/B cell studies Observation of fine ultrastructures of cells (100,000x magnification) Diopter rings: adjust for focusing differences between eyes Rubber eyeguard: adjust for comfort Eyepiece tube clamp screw: loosen to rotate head Reverse facing nosepiece: for ease in specimen manipulation Revolving nosepiece: use to rotate objectives Objectives: lenses w/c form primary image of specimen Field diaphragm: aperture diaphragm w/c restricts area of illumination Field diaphragm control ring: adjust size opening of field diaphragm Coarse focus knob: brings slide into view Fine focus knob: sharpens image Lamp socket: holds light source Interpupillary distance scale: indicates distance between eyes Eyepiece: magnify image formed by objective lens Stage: holds specimen Slide holder: holds slide in place Condenser control ring: adjusts size opening of condenser Condenser: aperture diaphragm that controls light Condenser centering screws: center the field of view Condenser focus know: focuses light onto slide X/Y travel knobs: moves slides on stage Brightness control dial: turns microscope on/off, adjusts light intensity TM = Ocular x Objective (10)(LPO: 10) = 100x (10)(HPO: 40) = 400x (10)(OIO: 100) = 1,000x Hemoglobin Determination AM, PM Strenuous muscular activity Altitude = Hgb Lying down Rgt: 0.1N HCl Comparing the brownish yellow color of solution to standard comparator block Rgt: 0.1N NaOH Not for newborns (HbF: alkali resistant) 1g Hgb = 1.34mL O2 1g Hgb = 3.47mg Fe2+ CuSO4 method (See BB notes) Manual and automated Dilution = 0.02mL blood: 5mL rgt (1:251) Reagents: Drabkin’s reagent (Brown container) Page | 211
Rule of three
Macromethods
Wintrobe tube
Micromethod (Adam)
a. Potassium ferricyanide = ferrous ferric b. Potassium cyanide = Hi HiCN c. Nonionic detergent = lysis of red cells, decreases turbidity d. Sodium bicarbonate (Orig. Drabkin’s) = result is read after 15mins e. Dihydrogen potassium phosphate (Mod. Drabkin’s) = result is read after 3 mins *Color intensity is measured at 540nm *All forms of Hgb are measured except SulfHb *Overanticoagulation does not affect result ♫ Turbidity = False a. High WBC count: to correct, centrifuge read supernatant b. HbS & HbC: to correct, dilute 1:1 w/ H2O result x 2 c. Lipemic blood: prepare patient’s blank (pt. plasma + HiCN rgt) 3 x RBC = Hgb 3 x Hgb = Hct Apply only to normocytic, normochromic red cells Hematocrit Determination Large volume of blood 1. Wintrobe & Landsberg = Double oxalate 2. Van Allen = Sodium oxalate 3. Haden = Sodium oxalate 4. Sanford-Magath = Sodium oxalate 5. Bray = Heparin Centrifuge: 2000-2300g for 30mins Layers (Spun Hct): 1st: Fatty layer = barely visible unless the patient is lipemic 2nd: Plasma 3rd: Buffy coat (1mm = 10,000 WBCs/mm3) Bottom: Packed cells Length = 11.5cm Bore = 3.0mm Calibration = 0-100 a. Left: 0-100 (ESR) b. Right: 100-0 (Hct) Capillary tube: Length = 7.0-7.5cm (70-75mm) Bore = 1mm Anticoagulant: Red (Heparin) No anticoagulant: Blue Centrifuge: 10,000-15,000g for 5mins Trapped plasma: plasma that remains in RBC portion after spinning = in poikilocytosis ♫ Advantages: a. Better packing of cells b. Less blood c. Less time consumed ♫ Layers: Top: Plasma 2nd: Platelets Page | 212
Automated methods
RBCs RBC diluting fluids
RBC count WBCs WBC Diluting fluids
WBC count Fuch’s Rosenthal
Speir’s Levy
Improved Neubauer
RBC count
3rd: Leukocytes 4th: Retics & nRBCs 5th: Mature RBCs Bottom: Clayseal (4-6mm) Hct = only computed Hct = MCV x RBC count RBC Count AM, PM Isotonic solutions 1.) NSS 2.) 3.8% Sodium citrate 3.) Dacies or formol citrate 4.) Hayem’s 5.) Toisson’s 6.) Bethell’s 7.) Gower’s Dilution (RBC pipette) = 0.5:100 (Blood: Diluent) = 1:200 RBC/mm3 = # RBC x AF x DCF x DF = # RBC x 5 x 10 x 200 = # RBC x 10,000 WBC Count AM, PM Hypotonic solutions: lyse non-nucleated RBCs 1.) 1-3% acetic acid 2.) 1% HCl 3.) Turk’s diluting fluid: Gentian violet + glacial acetic acid (solid at 17’C) + H 2O Mix = 3 mins (To allow lysis of RBCs) Dilution (WBC pipette) = 0.5:10 (Blood: Diluent) = 1:20 Leukocytosis = Use RBC pipette (1:100 or 1:200) WBC/mm3 = # WBC x AF x DCF x DF Counting Chamber 2 counting areas Each CA w/ 16 1mm2 squares Depth = 0.2mm Depth factor = 5 Volume = Area x Depth x # CA = 16mm2 x 0.2mm x 2 = 6.4mm3 Volume/counting chamber = 3.2mm3 4 counting areas Each CA w/ 10 1mm2 squares Depth = 0.2mm Depth factor = 5 Volume = Area x Depth x # CA = 10mm2 x 0.2mm x 4 = 8mm3 Volume/counting chamber = 2mm3 2 primary squares Each 1’ square w/ 9 1mm2 2’ squares Depth = 0.1mm Depth factor = 10 Volume = Area x Depth x # CA = 9mm2 x 0.1mm x 2 = 1.8mm3 Volume/counting chamber = 0.9mm3 Center square: Page | 213
WBC count Nucleated RBCs
w/ 25 3’ square Each 3’ square w/ 16 small squares 25 x 16 = 400 5 (counted) x 16 = 80 small squares 4 corners: Each 2’ square w/ 16 3’ squares 4 x 16 = 64 3’ squares Not lysed by WBC diluents Falsely counted as WBCs
NV: Adult = ≥5 nRBC/100 WBC differential Newborn = ≥10 nRBC/100 WBC differential Formula for WBC Corrected WBC = uncorrected WBCs x 100 correction 100 + NRBCs Preparation and Staining Procedures for the Blood Smear Techniques 1. Cover glass smear (Ehrlich) 2. Cover glass and slide (Beacom) 3. Wedge smear/Push/Spreader slide technique 4. Spun smear/Spun/Spinner’s technique = Automated: Hemaspinner 0 0 25 (30-40 ) Angle between 2 slides 22 x 22mm Square coverslip 2-3mm Drop of blood 0.25 inch (1cm) Distance of blood drop from the frosted edge of the slide 0.5 inch Smear terminates near the end of the slide (automated spreader) Buffy coat smear For patients w/ WBC count of <1 x 109/L Demonstration of LE cell Thick blood smear For blood parasites Methanol Fixative for blood and BM smears Toxic, causes permanent blindness Romanowsky’s stains Wright’s Giemsa = preferred stain for blood parasites Modified Wright’s-Giemsa Leishman Jenner May-Grunwald -----------------------------------------------------------------------------------------------------Contains: = Methylene blue (or Azure B - oxidized): basic = Eosin: acidic w/in 2-3 hours of specimen Time blood smears should be stained collection pH 6.8 Blood and bone marrow staining pH 7.2 Malarial parasite staining Excessively blue stain Thick films Prolonged staining time Inadequate washing Too high alkalinity of stain Page | 214
Excessively pink stain
Cross-sectional or crenellation method Longitudinal method Battlement method WBC Counting
PV patients
Anemic patients
Neutrophils Lymphocytes Monocytes Eosinophils Basophils Neutrophilia Eosinophilia
Lymphocytosis
Diluents tends to cause excessive basophilia Insufficient staining Prolonged washing time Mounting coverslips before they are dry Too high acidity of the stain Buffer may cause excessive acidophilia Blood film is moved from side to side WBCs are counted from the tail toward the head of the smear Near the tail on a horizontal edge: count 3 consecutive horizontal edge fields, count 2 fields towards the center of the smear, count 2 fields horizontally, count 2 fields vertically to the edge 100 cells = routine 50 cells = if patient WBC count <1.0 x 109/L 200 cells: = >10% eosinophils = >2% basophils = >11% monocytes = lymphocytes > neutrophils (except in children) Thinner smear: - smaller blood drop - slow spread - low angle Thicker smear: - larger blood drop - fast spread - increase angle Relative = 47-77% Absolute = 1.8-7.8 x 109/L Relative = 20-40% Absolute = 1.0-4.8 x 109/L Relative = 2-10% Absolute = 0.01-0.8 x 109/L Relative = 0-6% Absolute = 0-0.6 x 109/L Relative = 0-1% Absolute = 0-0.2 x 109/L Appendicitis Myelogenous leukemia Bacterial infections Allergies Scarlet fever Parasitic infections (T. spiralis) Eosinophilic leukemia Viral infections Whooping cough IM Lymphocytic leukemia Lymphoma Page | 215
Monocytosis
Shift to the left Shift to the right Reticulocyte count
% Retics Absolute reticulocyte count Corrected reticulocyte count Reticulocyte production index/Shift correction
Maturation time of retics in the blood RPI > 3
RPI < 2 Miller disk Eosinophil count Eosinophilia
Eosinopenia Eosinophil diluting fluids
Brucellosis Tuberculosis Monocytic leukemia SBE Typhoid Rickettsial infections Collagen disease Hodgkin’s disease Gaucher’s disease (+) immature granulocytic cells Leukemia Bacterial infections Hypersegmented neutrophils (≥6 lobes) NV: Adult = 0.5-1.5% (Ave: 1.0%) Newborn = 2-6% [# Retics ÷ # RBC (1000)] x 100 ARC = (% Retics ÷ 100) x RBC count (1012/L) x 1,000 NV = 25-75 x 109/L CRC = % Retics x (Patient Hct ÷ Normal Hct [0.45L/L]) NV = 1 General indicator of the rate of erythrocyte production increase above normal in anemias Indicates BM response to anemia RPI = CRC ÷ Maturation time of retics in the blood NV = 1 (Hct: 45%) 1.0 day = Hct: 45 ± 5% 1.5 days = Hct: 35 ± 5% 2.0 days = Hct: 25 ± 5% 2.5 days = Hct: 15 ± 5% Adequate response of BM to anemia - Chronic hemolysis - Recent hemorrhage - Response to therapy Inadequate response of BM to anemia - Aplastic anemia - Ineffective erythropoiesis (megaloblastic anemia) % Retics = Retics (A) ÷ [RBC (B) x 9] x 100 NV = 50-350 x 106/L Allergic reactions Parasitic infections Brucellosis Leukemias Hyperadrenalism (Cushing’s disease) Shock Administration of ACTH Composition: a. Phloxine/eosin/neutral red iodide = stains eosinophils Page | 216
Thorn’s test
RBC indices MCV MCH MCHC Defective centrifuge
MCHC >38% does not occur MCHC will not fall <22% ESR ESR
ESR
b. Propylene glycol = lyses RBCs c. Na2CO3 = lyses WBCs except eosinophils, intensifies staining of granules d. Heparin = prevents clumping Diluting fluids: 1. Pilot’s = phloxine 2. Manner’s = phloxine 3. Randolph’s = phloxine 4. Hinkleman = eosin 5. Tannen’s = neutral red iodide Assess adrenocortical function Sample 1: fasting specimen Sample 2: 4 hrs after administration of ACTH (eo. count) Normal: Eo. count: 1 > 2 (lower by 50%) Abnormal: Eo. count: 1 = 2 (hypoadrenalism) Erythrocyte Indices (Wintrobe Indices) Classify anemia according to RBC morphology MCV = (Hct ÷ RBC) x 10 NV = 80-100 fL (old: μm3) MCH = (Hgb ÷ RBC) x 10 NV = 27-32 pg (old: μμg) Rarely used MCHC = (Hgb ÷ Hct) x 100 NV = 31-36% (31-36 g/dL) Values affected: = Hct = MCV = MCHC Incorrect calculation (+) cold agglutinins Lipemia (+) HbS & HbC Erythrocyte Sedimentation Rate (ESR) Nonspecific measurement used to detect & monitor an inflammatory response to tissue injury Erythrocytes: *Macrocytes *Anemia Plasma composition: most important determinant *Fibrinogen *α1-globulin *α2-globulin *β-globulin *γ-globulin *Cholesterol Technical factor: *Tilting = 30 angle = 30% error *Temp. Erythrocytes: *Microcytes Page | 217
Stages of ESR
Wintrobe & Landsberg
Standard/Original Westergren
Modified Westergren Zeta Sedimentation Ratio (ZSR) Erythrocyte Osmotic Fragility test (Griffin and Sanford method)
Ascorbate cyanide screening
G6PD fluorescent screening Paroxysmal nocturnal hemoglobinuria (PNH) Sucrose hemolysis test
*Poikilocytes *Polycythemia *Anisocytes Plasma factor: most important determinant *Albumin *Lecithin Technical factor: *Overanticoagulation = EDTA = shrinkage of RBC = Hct, ESR 10 mins = 1. Initial rouleaux 40 mins = 2. Rapid settling of RBCs 10 mins = 3. Final sedimentation of RBCs 60 mins = Total Requires smaller amount of blood Involves no dilution Length: 11.5cm (115mm) Internal bore: 3.0mm Anticoagulant: Double oxalate Most sensitive Requires more blood Length: 300mm Internal bore: 2.65 ± 0.15mm Anticoagulant: Citrate (black) Anticoagulant-to-Blood ratio = 1:4 Anticoagulant: 2mL EDTA + 0.5mL NSS/Citrate Not affected by anemia Major disadvantage: requires special capillary tubes and Zetafuge ZSR = (%Hct ÷ %Zetacrit) x 100 Anticoagulant: Heparin % NaCl = # drops NaCl x 0.02 Add RBCs, stand for 2hrs at room temp Check for hemolysis (pink/red supernatant) NV: - Initial hemolysis = tube 21 or 22 (0.42-0.44%) - Complete hemolysis = tube 16 or 17 (0.32-0.34%) Detects deficiencies in the pentose phosphate pathway: - G6PD - glutathione peroxidase - glutathione reductase Rgts: - Na ascorbate - Na cyanide Normal = red (-) Enzyme = brown G6P + NADP ---(RBC: G6PD)---> 6-phosphogluconate + NADPH (fluorescence) Normal: Max fluorescence at 10mins G6PD def: Little or no fluorescence Acquired disorder in w/c red cells are abnormally sensitive to complement (-) DAF Screening test for PNH Page | 218
Ham’s acidified serum test
Patient has received normal RBCs PCH
Patient RBCs + ABO compatible serum + sucrose solution Normal = (-) Hemolysis PNH = (+) Hemolysis Confirmatory test for PNH Tube 1: Patient RBCs + normal serum + weak acid (0.2N HCl) Tube 2: Patient RBCs + patient serum + weak acid (0.2N HCl) Tube 3: Patient RBCs + normal inactivated serum + weak acid (0.2N HCl) Normal = (-) Hemolysis on all tubes PNH = (+) Hemolysis except on Tube 3 (inactivated serum) Patient w/ PNH + blood transfusion ---(Ham’s test)---> Hemolysis
IgG autoanti-P = biphasic hemolysin - Cold = attaches to RBCs - Warm = RBC lysis Donath-Landsteiner test Test for PCH Ctrl: Patient WB incubate at 37’C for 30mins incubate at 37’C for 30mins Test: Patient WB incubate at 4’C for 30mins incubate at 37’C for 30mins Normal = (-) hemolysis on test and control PCH = (-) hemolysis on control but (+) hemolysis on test sample Autohemolysis test Blood alone ---(48 hrs)---> Hemolysis: >0.2 to 2% Blood + glucose ---> Hemolysis: 0-0.8% Blood + ADP ---> Hemolysis: 0-0.9% -----------------------------------------------------------------------------------------------------G6PD deficiency (PPP) = corrects w/ glucose only PK deficiency (EMP) = corrects w/ ADP only H. Spherocytosis = corrects w/ ADP and glucose Potential Causes of Erroneous Results with Automated Cell Counters Parameter Causes of Spurious Increase Causes of Spurious Decrease WBC count Cryoglobulin Clotting Cryofibrinogen Smudge cells Heparin Uremia plus immunosuppressants Monoclonal proteins Nucleated RBCs Platelet clumping Unlysed RBCs Platelet count Cryoglobulin Clotting Cryofibrinogen Giant platelets Hemolysis Heparin Microcytic RBCs Platelet clumping RBC inclusions Platelet satellitism WBC fragments RBC count Cryoglobulin Autoagglutination Cryofibrinogen Clotting Giant platelets Hemolysis WBC >50,000/μL Microcytic RBCs Hemoglobin HbCO >10% Clotting Cryoglobulin Sulfhemoglobin Cryofibrinogen Hemolysis Page | 219
Hematocrit (automated)
Hematocrit (microhct)
Heparin WBC >50,000/μL Hyperbilirubinemia Lipemia Monoclonal proteins Cryoglobulin Cryofibrinogen Giant platelets WBC >50,000/μL Hyperglycemia >600mg/dL Hyponatremia Plasma trapping
Autoagglutination Clotting Hemolysis Microcytic RBCs Excess EDTA Hemolysis Hypernatremia Cryoglobulin Cryofibrinogen Giant platelets Hemolysis Microcytic RBCs Swollen RBCs WBC >50,000/μL Spuriously low Hgb Spuriously high Hct
MCV
Autoagglutination WBC >50,000/μL Hyperglycemia Reduced red cell deformability
MCHC
Autoagglutination Clotting Hemolysis Spuriously high Hgb Spuriously low Hct Increased: WBC count, RBC count, Platelet count, Hgb, Hct Decreased: MCV Increased: WBC count, Hgb Decreased: Platelet count
Cryoglobulin Cryofibrinogen Heparin Clotting Hemolysis Autoagglutination Defibrinated blood
Optical light scattering
Electrical impedance
Increased: MCHC Decreased: WBC count, RBC count, Platelet count, Hgb, Hct Increased: Hgb, MCHC, Platelet count Decreased: RBC count, Hct, MCV Increased: MCV, MCHC Decreased: RBC count, Hct Blood Glass Beads/clips Tests: “OAA” - OFT - Autohemolysis test - Acidified serum test Automated Cell Counter Blood cells when subject to light will create forward & side light scatters w/c are detected by photodetector Forward LS = cell size Side LS/900/right angle scatter = cell granularity Ex. Technicon autoanalyzer Blood cells are nonconductors of electricity. they create impedance or resistance of current when passed in a solution that conduct electricity Ex. Sysmex counter, Coulter counter Page | 220
Coulter counter
Histograms
Ohm’s law
Positive error
Negative error Polychromasia grading
Normocytic, Normochromic RBCs
Hemolytic anemias
Triplicate count (3x) a. Blood is diluted 1:6250 (isotonic) ♫ RBCs = 36-360fL ♫ Plts = 2-20fL b. Blood is diluted 1:251 (hypotonic) ♫ Lymphocytes = 35-90fL ♫ Monocytes = 90-160fL ♫ Granulocytes = 160-450fL RBCs, WBCs, plts X-axis - Horizontal/abscissa - Size of cells Y-axis - Vertical/ordinate - Number of cells V=IxR Where: V = voltage I = current R = resistance Count: “BEA” ♫ Bubbles ♫ Extraneous electrical pulses ♫ Aperture plug Count ♫ Improper setting of aperture error % of RBCs that are polychromatophilic Slight = 1% 1+ = 3% 2+ = 5% 3+ = 10% 4+ = >11% 1. Defective formation of RBCs or the presence of tumor cells in BM: *Aplastic anemia *Leukemia *Hodgkin’s disease *Multiple myeloma *Leukoerythroblastosis *Metastatic cancer *Anemia of renal & endocrine disease *Anemia of inflammatory disease 2. Abnormal hemoglobin, increased destruction of RBCs *Certain acquired hemolytic anemia *PNH *Sickle cell anemia *HDN *Anemia of chronic renal insufficiency 1. Intrinsic defects w/in RBC a. Hereditary – membrane defects Page | 221
**Spherocytosis **Elliptocytosis **Acanthocytosis **Stomatocytosis **Rh null disease b. Hereditary – enzyme defects **G6PD **PK c. Hereditary – hemoglobinopathies **Sickle cell disease **Hemoglobin C disease d. Unstable hemoglobin disease **Hemoglobin E disease e. Hereditary – defective globin synthesis **Thalassemia f. Acquired **PNH 2. Extracorpuscular causes: nonimmune acquired hemolytic anemias *Chemicals, toxins, venoms *Physical trauma: disorders causing fragmentation (burns, cardiac replacement valves, MAHA, HUS) 3. Extracorpuscular causes: immune hemolytic anemias *Isoimmune antibodies: incompatible blood transfusion, HDN *Autoimmune antibodies: warm/cold reacting, drug-induced 4. Miscellaneous *Anemia of liver disease *Sulfhemoglobinemia *Porphyrias *Methemoglobinemias
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