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Hematology Greek:
-Haima = Blood
-Logos = Study/science
EDTA Chelates calcium
(Lavender top) 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
Modified Westergren ESR 2mL EDTA + 0.5mL NSS/Citrate
(Black top tube) Ratio = 1:4 (Anticoagulant-to-Blood)
Citrate For coagulation and platelet studies
(Light blue top tube) = Preserves labile factors V and VIII
= Buffered 3.2% (0.109M) citrate
Inversion: 3-4x
Ratio = 1:9 (Anticoagulant-to-Blood)
Polycythemic patients Hct
Excess Citrate = PT, APTT
Remedy: Reduce the volume of citrate
Amount of citrate = [(100-Hct)÷(595-Hct)] x mL WB
Oxalate Double/balanced oxalate (Ratio = 2:3): Maintained cell structures
a. Potassium oxalate (Paul-Heller’s) = shrink cells
b. Ammonium oxalate (Wintrobe’s) = swell cells
Heparin 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
Order of Draw Evacuated tube:
(Henry 21st Edition) 1. Sterile blood culture tube
2. Citrate (blue)
3. Nonadditive tube (red)
4. Heparin (green)
5. EDTA (lavender)
6. Fluoride (gray)
Order of Draw 1. EDTA
(Syringe method) 2. Other anticoagulated tubes
3. Nonadditive tube
EDTA containing tubes Lavender
Pink
White
Royal blue
Tan
Skin puncture 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:
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WBCs
Hgb, Hct, RBCs, platelet
Venipuncture Veins in the arms (antecubital region):
1. Median cubital = preferred, most stable
2. Cephalic (lateral)
3. Basilic (medial)
Common gauge (needle) 19, 20, 21
Routine: 20g
Common length of needle 1-1.5 inches
Color coded hub (gauge) 18 = pink
21 = green
22 = gray
23 = blue/light blue/turquoise
25= orange
Angle Venipuncture: 150
BB: 450 10-200 once in the skin
Tourniquet 3-4 inches above the site (7.5-10cm)
Not exceed 1min/2mins
Prolonged application hemoconcentration
BP cuff as tourniquet 40-60 mmHg
Reassure the patient Crying = cell count
Position the patient Lying down = hemodilution ( PCV by 8%, WBC)
Lying up = hemoconcentration
IV line 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
Hematopoiesis Cellular formation, proliferation, differentiation and maturation of blood cells
Mesoblastic period 19th day of gestation
Yolk salk = Erythropoiesis
Embryonic hemoglobins:
a. Gower 1 = Zeta2 + Epsilon2
b. Portland = Zeta2 + Gamma2
c. Gower 2 = Alpha2 + Epsilon2
Hepatic period 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
Myeloid period Between 5th & 6th month of gestation persist throughout life
BM = 1’ source of cell production (Hematopoiesis)
Sternum = principal source of hematopoiesis in adults
Adults HbA1 = ≥95%
HbA2 = 1.5-3%
HbF = <2%
Neonates HbF = 60-80%
HbA = 20-40%
Marrow specimens 1. Trephine (Core) Biopsy
= Trephine biopsy needle (Jamshidi needle)
2. Aspiration
= Aspiration needle (University of Illinois sterna needle)
Posterior iliac crest Safest site for BM aspirate/biopsy
M:E ratio 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
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Leukemia = 25:1
Neutrophilic, Eosinophilic, Basophilic precursors = Myeloid
Erythroid precursors
Monocytic precursors = not included
BM Cellularity Percentage of marrow space occupied by hematopoietic cells compared w/ fat
Normocellular marrow (Adult): 30% - 70% hematopoietic cells
♫Fat = 10-50%
♫Hematopoietic elements = 40-60% (Ave. 50%)
Yellow BM Fats
Red BM Hematopoietic cells
Marrow differential Recommended that at least 500, preferably 1000 cells be counted for a marrow
differential
Metamyelocyte/Juvenile Predominant cell (WBC) in adult BM (up to 32%)
granulocyte
Stem cells <1% cells in BM
Osteoblasts Bone forming cells
Confused w/ plasma cells
Waterbug or comet appearance
Osteoclasts Bone destroying cells
Confused w/ megakaryocytes
CD2, CD3 T cells
CD19, CD20 B cells
CD34 Stem cell marker (lymphoid and myeloid precursor)
CD16, CD56 NK cells
CD10 CALLA (Common ALL Antigen)
Erythropoietin Produced by the kidney
Primary regulator of erythropoiesis
Thrombopoietin Produced by kidney and liver
Regulator of thrombopoiesis
Erythropoiesis
Erythropoiesis 1’ stimulus = Hypoxia
IL-3 Multi-CSF (Colony Stimulating Factor)
Stimulates hematopoietic cells
1. Pronormoblast Proerythroblast/rubriblast
N/C ratio = 8:1
Nucleoli = 1-2
Can produce up to 16 RBCs per 1 pronormoblast/rubriblast
2. Basophilic normoblast Prorubricyte
Intensely basophilic cytoplasm
N/C ratio = 6:1
Nucleoli usually not visible
3. Polychromatophilic Rubricyte
normoblast 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
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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
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
c. Basophil myelocyte = dark purple or blue-black granules
Last stage capable of mitosis
1st: Secondary granules
N/C ratio = 1:1
(-) Nucleoli
4. Metamyelocyte Juvenile granulocyte
Not capable of mitosis (post-mitotic pool)
Indented/kidney-shaped nucleus
Predominant WBC in BM
5. Band 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
6a. Segmented neutrophil Rose-pink granules
Nucleus: 2-5 lobes
Diurnal variation (PM)
Specific granules:
a. Lysozyme
b. Lactoferrin
c. Collagenase
d. Plasminogen activator
e. Aminopeptidase
6b. Eosinophil 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
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e. Eosinophil protein X
f. Phospholipase
[Smaller]
a. Arylsulfatase
b. Acid phosphatase
6c. Basophil 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
Monopoiesis
Monopoiesis 1. Monoblast
2. Promonocyte
3. Monocyte
Monocyte 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)
Lymphopoiesis
Lymphopoiesis 1. Lymphoblast
2. Prolymphocyte
3. Lymphocyte
Lymphocyte Small = 8-10μm (Size = RBC)
Medium = 10-12μm
Large = 12-16μm (Rare)
Cytoplasm: bluish (Robin’s egg blue)
Nucleus: compact
T lymphocytes 60-80%
Long-lived (4-10 years)
B lymphocytes 10-20%
Short-lived (3-4 days)
Can differentiate into plasma cell or memory B cells
Null lymphocytes Large granular lymphocyte
10%
Plasma cells differentiation 1. Plasmablast
2. Proplasmacyte
3. Plasmacyte/Plasma cell
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”
Thrombopoiesis
Thrombopoiesis 5 days (Megakaryoblast Platelets)
1. Megakaryoblast N/C ratio = 10:1
2. Promegakaryocyte Nucleus: may show slight lobulation (Endomitosis)
N/C ratio = 4:1 to 7:1
3. Granular megakaryocyte
4. Mature megakaryocyte 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
5. Metamegakaryocyte Disintegrated cell surrounded by platelet
6. Platelet/Thrombocyte 1-4μm in diameter
Light blue to purple
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Very granular
a. Chromomere: granular and centrally located
b. Hyalomere: surrounds the chromomere, nongranular and clear to light blue
Life span: 8-11 days
2/3 (67%) Circulating platelets
1/3 (33%) Platelets stored in the spleen
Endomitosis Nuclear division w/o cytoplasmic division
2000-4000 # of platelets a megakaryocyte can produce
1 heme molecule 1 mol O2
1 hemoglobin 4 mol O2
Mitochondria Early and late heme synthesis
141 amino acids Alpha
146 amino acids Beta, Gamma, Delta, Epsilon, Zeta
Chromosome 11 Alpha, Zeta
Chromosome 16 Beta, Gamma, Delta, Epsilon
Oxyhemoglobin Normal = Sigmoid in shape
Dissociation Curve X-axis = Hgb concentration in g/dL | Y-axis = OD
Shift to the left (ODC) CO2
Temperature
2,3-DPG
pH
Affinity of Hgb for O2
HbF
Shift to the right (ODC) CO2
Temperature
2,3-DPG
pH
Affinity of Hgb for O2
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Cytoplasmic Abnormalities
Alder-Reilly granules 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)
Toxic granules Large purple to black granules resembling ALR granules
♫ Infections
♫ Toxic states
Toxic vacuoles Infections
Toxic states
Auer rods Pink or red rod shaped structures
Fused primary granules (peroxidase positive)
Myeloid and monocytic series only
Faggot cells w/ mass of Auer rods
M3 (APL) = associated w/ DIC
Chediak-Higashi granules 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)
May-Hegglin inclusion Pale blue inclusions derived from RNA
♫ May-Hegglin anomaly
= Autosomal Recessive
= Giant platelets
= Thrombocytopenia
Resemble Dohle bodies (IT)
Dohle bodies Single or multiple blue inclusions
Dohle-Amato bodies Aggregates of free ribosomes of rough ER
Resembles
♫ Infections
♫ Toxic states
IT: Infections, Toxic states Dohle bodies
Toxic granules
Toxic vacuoles
Abnormalities in Function
Job’s syndrome Normal random activity
Abnormal chemotactic activity
Lazy leukocyte syndrome Abnormal random and chemotactic activity
Chronic Granulomatous Disease Inability of phagocytes to kill ingested microorganisms
(CGD) Impaired NADPH oxidase
Impaired oxidative metabolism/respiratory burst
Test: NBT dye test
Cells Exhibiting Phagocytosis
LE cell Neutrophil w/ large purple homogeneous round inclusion
Believe to be a neutrophil that ingested another neutrophil
Buffy coat
Smooth and evenly stained
♫ SLE
Tart cell Monocyte w/ ingested lymphocyte
Rough and unevenly stained
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HEMOSTASIS
Primary hemostasis Involves blood vessels and platelets
Formation of platelet plug
Test: Bleeding time
Platelets Functions:
Adhesion
Activation
Release
Aggregation
Secondary hemostasis Involves the coagulation factors
Formation of stable fibrin clot
Test: Clotting time
Arteries Carry blood from the heart to the capillaries
Primary Hemostasis
Veins Return blood from the capillaries to the heart
Capillaries Injured vessel: vasoconstriction
= initiated by serotonin and thromboxane A2 derived from platelets and
endothelial cells
Maturation Stage Cytoplasmic Cytoplasmic Tags Nuclear Features Thrombocytes
Granules Visible
Megakaryoblast (-) (+) Single nucleus (-)
Fine chromatin
(+) Nucleoli
Promegakaryocytes Few (+) 2 nucleus (-)
Megakaryocytes Numerous Usually (-) 2 or more nuclei (-)
Metamegakaryocytes Aggregated (-) 4 or more nuclei (+)
Platelet structure 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 Adhesion Platelet adherence to exposed subendothelial surface (collagen)
Occurs in the presence of von Willebrand factor
In vivo: collagen
In vitro: glass
Bernard-Soulier syndrome (-) gpIb = receptor for vWF
(Giant platelet syndrome)
Von Willebrand disease (-) vWF = for platelet adhesion
Platelet Activation Morphologic and functional changes in platelets
Agonists: substance that initiate activation
Arachidonic acid ---(Cyclooxygenase)---> Thromboxane A2
TxA2 Vasoconstrictor
Stimulate platelet secretion
Aspirin Inhibits COX
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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)
Secondary Hemostasis
Coagulation factors All are produced in the liver except Factor VIII
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
Numeral Preferred Name Synonyms
I Fibrinogen
II Prothrombin Prethrombin
III Tissue factor Tissue thromboplastin
IV Calcium
V Proaccelerin Labile factor
Accelerator globulin (aCg)
VII Proconvertin Stable factor
Serum prothrombin conversion
accelerator (SPCA)
VIII:C Antihemophilic factor Antihemophilic factor globulin (AHG)
Antihemophilic factor A
Platelet cofactor 1
IX Plasma thromboplastin component Christmas factor
Antihemophilic factor B
Platelet cofactor 2
X Stuart-Prower factor Stuart factor
Prower factor
Autoprothrombin III
XI Plasma thromboplastin component Antihemophilic factor C
XII Hageman factor Glass factor
Contact factor
XIII 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
Stage I: Generation of Intrinsic: XIIXIIX—VIII (Cofactor)
Thromboplastin Extrinsic: IIIVII
Common: X—V (Cofactor)
----> Common thromboplastin/Prothrombinase (Va-Xa-Ca2+-PL)
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Stage II: Conversion of II ---(Prothrombinase)---> Thrombin
prothrombin to thrombin
Stage III: Conversion of I ---(Thrombin)---> Fibrin clot
fibrinogen to fibrin clot XIII---(Thrombin)---> XIIIa
Fibrin clot ---(XIIIa)---> Stable fibrin clot
Fibrinogen Most concentrated
Calcium Involved in all stages of coagulation except contact phase
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
Fresh Plasma Aged Plasma Adsorbed Plasma Fresh Serum Aged Serum
I + + + - -
II + + - + (<20%) -
V + - + - -
VII + + - + +
VIII + - + - -
IX + + - + +
X + + - + +
XI + + + + +
XII + + + + +
XIII + + + - -
Prothrombin:
80% is consumed during coagulation
<20% residual prothrombin
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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
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HEMATOLOGY PROCEDURES
Brightfield microscopy Examine blood films
Oil immersion microscopy Erythrocyte morphology & leukocyte differential
Phase-contrast microscopy For manual platelet counts
Fluorescence microscopy ANA and T/B cell studies
Electron microscopy Observation of fine ultrastructures of cells (100,000x magnification)
Basic component of Diopter rings: adjust for focusing differences between eyes
Standard light microscope 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
Hemoglobin Determination
Hemoglobin AM, PM
Strenuous muscular activity
Altitude = Hgb
Lying down
Acid hematin Rgt: 0.1N HCl
Comparing the brownish yellow color of solution to standard comparator block
Alkali hematin Rgt: 0.1N NaOH
Not for newborns (HbF: alkali resistant)
Gasometric (Van Slyke) 1g Hgb = 1.34mL O2
Chemical 1g Hgb = 3.47mg Fe2+
(Kennedy’s, Wong’s)
SG method CuSO4 method (See BB notes)
Cyanmethemoglobin/ Manual and automated
Hemiglobincyanide method Dilution = 0.02mL blood: 5mL rgt (1:251)
Reagents:
Drabkin’s reagent (Brown container)
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
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*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)
Rule of three 3 x RBC = Hgb
3 x Hgb = Hct
Apply only to normocytic, normochromic red cells
Hematocrit Determination
Macromethods 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
Wintrobe tube Length = 11.5cm
Bore = 3.0mm
Calibration = 0-100
a. Left: 0-100 (ESR)
b. Right: 100-0 (Hct)
Micromethod (Adam) 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
3rd: Leukocytes
4th: Retics & nRBCs
5th: Mature RBCs
Bottom: Clayseal (4-6mm)
Automated methods Hct = only computed
Hct = MCV x RBC count
RBC Count
RBCs AM, PM
RBC diluting fluids Isotonic solutions
1.) NSS
2.) 3.8% Sodium citrate
3.) Dacies or formol citrate
4.) Hayem’s
5.) Toisson’s
6.) Bethell’s
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7.) Gower’s
Dilution (RBC pipette) = 0.5:100 (Blood: Diluent) = 1:200
RBC count RBC/mm3 = # RBC x AF x DCF x DF = # RBC x 5 x 10 x 200 = # RBC x 10,000
WBC Count
WBCs AM, PM
WBC Diluting fluids 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) + H2O
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 count WBC/mm3 = # WBC x AF x DCF x DF
Counting Chamber
Fuch’s Rosenthal 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
Speir’s Levy 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
Improved Neubauer 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
RBC count Center square:
w/ 25 3’ square
Each 3’ square w/ 16 small squares
25 x 16 = 400
5 (counted) x 16 = 80 small squares
WBC count 4 corners:
Each 2’ square w/ 16 3’ squares
4 x 16 = 64 3’ squares
Nucleated RBCs 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
X-axis
- Horizontal/abscissa
- Size of cells
Y-axis
- Vertical/ordinate
- Number of cells
Ohm’s law V=IxR
Where:
V = voltage
I = current
R = resistance
Positive error Count: “BEA”
♫ Bubbles
♫ Extraneous electrical pulses
♫ Aperture plug
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Negative error Count
♫ Improper setting of aperture error
Polychromasia grading % of RBCs that are polychromatophilic
Slight = 1%
1+ = 3%
2+ = 5%
3+ = 10%
4+ = >11%
Normocytic, 1. Defective formation of RBCs or the presence of tumor cells in BM:
Normochromic RBCs *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
Hemolytic anemias 1. Intrinsic defects w/in RBC
a. Hereditary – membrane defects
**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. Intracorpuscular 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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