Professional Documents
Culture Documents
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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:
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
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
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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 normoblast Metarubricyte
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-
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
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
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3. Lymphocyte
Lymphocyte Small = 8-
Medium = 10-
Large = 12- (Rare)
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])
(ALA synthase)
D-Aminolevulinic acid
(ALA dehydrase)
Porphobilinogen
(Uroporphyrinogen synthase)
(Uroporphyrinogen cosynthase)
Uroporphyrinogen
(Uroporphyrinogen decarboxylase)
Coproporphyrinogen
(Coproporphyrinogen oxidase)
Protoporphyrinogen
(Protoporphyrinogen oxidase)
Protoporphyrin IX + Fe2+
(Ferrocheletase)
HEME
Arterial blood O2 saturation = 95%
pO2 = 95 mmHg
Venous blood O2 saturation = 70%
pO2 = 40 mmHg
P50 pO2 = 26.6 mmHg
Bohr effect pH = Hgb affinity for O2 --- (L)
pH = Hgb affinity for O2 --- (R)
Oxyhemoglobin (HbO2) Arterial blood
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Unconjugated bilirubin
Urine and fecal urobilinogen
Intravascular hemolysis 10% aged red cell destruction
w/in the blood vessels
Haptoglobin
Hemopexin
(+) Hemoglobinemia
Anisocytosis Variation in size
RDW Numerical expression that correlates w/ the degree of anisocytosis
NV = 11.5-14.5%
Anisochromia Variation in Hgb content (Ex. Dimorphic anemia)
MCV NV = 80-100fL (Normocytic)
<80fL = Microcytic
>100fL = Macrocytic
MCH NV = 27-32 pg
MCHC NV = 31-36% (Normochromic)
<31% = Hypochromic
>36% = Hyperchromic
Normocytic normochromic 1. Acute blood loss
anemia 2. Hemolytic anemia
3. Aplastic anemia
Microcytic hypochromic 1. Chronic blood loss = most common cause
anemia 2. Thalassemia = N-RDW
3. IDA = Iron, TIBC (Ex. Hookworm infections) | RDW
4. Sideroblastic anemia
5. Chronic disease
Macrocytic, normochromic 1. Megaloblastic anemia = Vitamin B12/Folate deficiency
anemia 2. Nonmegaloblastic anemia = Liver disease, alcoholism
Aplastic anemia
Acquired = radiation, chemical (benzene), drugs (chloramphenicol)
Pancytopenia = WBCs, RBCs, Retics Plts
TIBC Differentiates IDA (TIBC) from other microcytic, hypochromic anemia
Degree of Hypochromia 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
Megaloblastic anemia Oval macrocytes
Howell-Jolly bodies
Hypersegmented neutrophils
Ineffective erythropoiesis (pancytopenia)
Vitamin B12 (Cobalamin) w/ CNS problems
deficiency 1. Pernicious anemia
= Deficient in intrinsic factor (produced by parietal cells) for B12 absorption
2. D. latum infection
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3. Vegetarian diet
4. Malabsorption syndrome
= Steatorrhea, sprue
Folic acid (Vit. B9) deficiency w/o CNS problems
1. Pregnancy
2. Dietary deficiency
3. Steatorrhea, sprue
Polychromasia Reticulocytosis
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Semilunar bodies Large, pale pink staining ghost of the red cell
Half-moon cell Membrane remaining after the contents have been released
Crescent cell Malaria
Burns Spherocytes
Schistocytes
Microspherocytes
Drepanocytes Crescent-shaped cell
Sickle cells Holly-leaf
Menisocytes Polymerization of deoxygenated Hgb
Sickle cell anemia
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HbA2 -thalassemia
Quantitation: Anion exchange microchromatography
HbF 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
Tests for unstable Hgb (HbH)
2. Isopropanol precipitation test: 17% solution
Sample Criteria for Erythrocyte Morphology Evaluation
Morphology Characteristics w/in Normal 1+ 2+ 3+ 4+
Limits (OIO) (per OIO) (per OIO) (per OIO) (per OIO)
0-5 5-10 10-20 20-50 >50
0-5 5-10 10-20 20-50 >50
Hypochromia 0-2 3-10 10-50 50-75 >75
Poikilocytosis 0-2 3-10 10-20 20-50 >50
Polychromatophilia -- 1-5 6-10 >10 --
Rouleaux -- Numerous --
Agg. of 3-4 RBCs Agg. of 5-10 RBCs
aggregates
Nuclear Abnormalities
Pelger-Huet Hyposegmentation (neutrophil)
Bilobed nucleus: Dumb-bell shaped/spectacle/peanut- -
Resembles Stab cell (To differentiate: PH cell has more clumped chromatin)
Pelger-Huet anomaly = Autosomal Dominant
Pseudo-Pelger-Huet = Acquired in myeloproliferative disorders
Hypersegmentation
Abnormal DNA synthesis
Undritz anomaly = hereditary hypersegmentation
Megaloblastic anemia
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
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Minimum of 1 or 2 years
Predominantly mature cells
Leukemic leukemia
Subleukemic leukemia
(+) Abnormal and immature cells in PB
Aleukemic leukemia
(-) Abnormal and immature cells in PB
French-American-British Divides acute leukemias into lymphoblastic and monoblastic
(FAB) Classification of Acute = Subdivided according to cellular morphology, cytochemical staining results,
Leukemias cytogenetic studies and T & B lymphocytes marker results
Acute leukemia Normocytic, normochromic RBCs
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>30% blasts
>50% erythrocytic precursors
M7 Acute megakaryocytic leukemia
>30% blasts
>30% megakaryocytic cells
Chronic Myeloproliferative Disorders
MPD Proliferation of abnormal pluripotential stem cell
Stem cell differentiates into the granulocytic (myeloid stem cell), megakaryocytic
and erythroid cell lines
1. Chronic Myelogenous (+) Philadelphia chromosome: t(9+;22-) - both long arms
Leukemia (CML) If (-
Similar to leukomoid reaction, to differentiate:
a. Chromosome studies
b. LAP = ( in Leukomoid reaction, in CML)
2. Myelofibrosis w/ myeloid Fibrosis and granulocytic hyperplasia of BM, w/ granulocytic and megakaryocytic
metaplasia (MMM) 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
Other names: Polycythemia Vera, Polycythemia Rubravera, Vaquez Osler disease,
Panmyelosis
RBC mass ( Hct)
RBCs, WBCs, Platelets
Erythropoietin (EPO)
In response to hypoxia
appropriate production of In patients w/ pulmonary/cardiac disease
EPO RBCs, WBCs, Platelets
EPO
In patients w/ tumors of kidney, liver, brain, adrenal and pituitary gland
inappropriate production of RBCs, N-WBCs, N-Platelets
EPO EPO
Relative polycythemia Spurious/Gaisböck polycythemia
Associated w/ stress and anxiety
N-RBC mass
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CGD = 0 to negligible
Infection = 70%
Modified NBT w/ stimulating agent
Test:
Buffy coat (WBC) + Bacterial suspension ---(NBT)---> (+) Formazan
NV = 80-100% formazan
CGD = <50% formazan
Feulgen reaction Specific reaction for DNA
Howell-Jolly bodies = Feulgen (+)
Prussian blue stain Stain for hemosiderin
(+) Sideroblast
(+) Siderocytes
Supravital stain (RHH) RHH = NCB
Reticulum = New methylene blue
Heinz bodies = Crystal violet
Hemoglobin H = Brilliant cresyl blue
BCB Stain for automated cell counter
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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)
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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)
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
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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
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
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 Liver disease
Labile factor deficiency DIC
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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
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b. Ellagic acid
c. Celite
d. Kaolin
2. Phospholipids: substitute for platelets
3. CaCl2
Begin timing for clot formation on the addition of CaCl2 rgt
NV = 25-35 secs
Stypven time/Russel viper East Indian viper venom Vipera russelli: directly activates factor X
venom time Detect coagulation factor deficiencies involving common pathway
PPP + Stypven rgt: platelin-CaCl2 rgt
NV = 6-10 secs
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Automated instruments:
- Ortho Koagulab 16S and 40A
- Coag-A-Mate X2 and XC
- MLA Electra 700 and 800
Fibrinolysis Digestion of fibrin clot
Occurs when plasminogen plasmin
Plasminogen activators 1. Intrinsic activators
- XIIa
- Kallikrein
- HMWK
2. Tissue type
- Urokinase-like PA
3. Therapeutic activators = treatment for thromboemboli
- Streptokinase
- Urokinase
- Tissue-like PA
Inhibitors of fibrinolysis 2-antiplasmin = Major inhibitor
2-macroglobulin
Thrombospondin
PA inhibitor 1 and 2
Degradation of cross-linked Crosslinked fibrin (urea insoluble)
fibrin by plasmin
(Plasmin)
Fragment X
(Plasmin)
Fragment Y | Fragment D
(Plasmin)
Fragment D | Fragment D
Primary fibrinolysis 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
Secondary fibrinolysis 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
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Neoplasm
Snake bite
HTR
Laboratory Evaluation of Fibrinolysis
Whole blood clot lysis time
(WBCLT) Clot lysis prior to 48 hrs indicates excessive systemic fibrinolysis
Euglobulin lysis time 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
Protamine sulfate test -DIC) in the plasma
Plasma + Protamine sulfate + ETOH (+) gel-like clot (paracoagulation)
Ethanol gelation test -DIC) in the plasma
Plasma + NaOH (pH) + ETOH (+) pptn/gel
Latex D-dimer assay Most specific test for DIC
Test Primary Fibrinolysis Secondary Fibrinolysis
WBCLT < 48 hrs < 48 hrs
Euglobulin clot lysis < 2 hrs < 2 hrs
Ethanol gelation - +
Protamine sulfate - +
D-dimer - +
Heparin Injected
Action: inhibits thrombin
Monitoring: APTT = sensitive, method of choice (CAP)
Neutralize w/ protamine sulfate
Warfarin Oral
Coumarin WARF: Wisconsin Alumni Research Foundation
Coumadin Action: Vit. K antagonist, inhibits II, VII, IX, X
Monitoring: PT (reported in INR)
Neutralize w/ Vitamin K, FFP
INR 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
ISI International Sensitivity Index (PT rgt)
PT rgt is calibrated w/ Manchester rgt = from human brain thromboplastin
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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 Standard Diopter rings: adjust for focusing differences between eyes
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
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RBC Count
RBCs AM, PM
RBC diluting fluids Isotonic solutions
1.) NSS
2.) 3.8% Sodium citrate
3.) Dacies or formol citrate
4.)
7.)
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
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 count 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
Improved Neubauer 2 primary squares
2
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:
Each
25 x 16 = 400
5 (counted) x 16 = 80 small squares
WBC count 4 corners:
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NV:
differential
crenellation method
Longitudinal method WBCs are counted from the tail toward the head of the smear
Battlement method 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
WBC Counting 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)
PV patients Thinner smear:
- smaller blood drop
- slow spread
- low angle
Anemic patients Thicker smear:
- larger blood drop
- fast spread
- increase angle
Neutrophils Relative = 47-77%
Absolute = 1.8-7.8 x 109/L
Lymphocytes Relative = 20-40%
Absolute = 1.0-4.8 x 109/L
Monocytes Relative = 2-10%
Absolute = 0.01-0.8 x 109/L
Eosinophils Relative = 0-6%
Absolute = 0-0.6 x 109/L
Basophils Relative = 0-1%
Absolute = 0-0.2 x 109/L
Neutrophilia Appendicitis
Myelogenous leukemia
Bacterial infections
Eosinophilia Allergies
Scarlet fever
Parasitic infections (T. spiralis)
Eosinophilic leukemia
Lymphocytosis Viral infections
Whooping cough
IM
Lymphocytic leukemia
Lymphoma
Monocytosis Brucellosis
Tuberculosis
Monocytic leukemia
SBE
Typhoid
Rickettsial infections
Collagen disease
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4. Hinkleman = eosin
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Hyperbilirubinemia
Lipemia
Monoclonal proteins
Hematocrit (automated) Cryoglobulin Autoagglutination
Cryofibrinogen Clotting
Giant platelets Hemolysis
Microcytic RBCs
Hyperglycemia >600mg/dL
Hematocrit (microhct) Hyponatremia Excess EDTA
Plasma trapping Hemolysis
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Hypernatremia
MCV Autoagglutination Cryoglobulin
Cryofibrinogen
Hyperglycemia Giant platelets
Reduced red cell deformability Hemolysis
Microcytic RBCs
Swollen RBCs
MCHC Autoagglutination
Clotting Spuriously low Hgb
Hemolysis Spuriously high Hct
Spuriously high Hgb
Spuriously low Hct
Cryoglobulin Increased: WBC count, RBC count, Platelet count, Hgb, Hct
Cryofibrinogen Decreased: MCV
Heparin Increased: WBC count, Hgb
Decreased: Platelet count
- OFT
- Autohemolysis test
- Acidified serum test
Automated Cell Counter
Optical light scattering 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
Electrical impedance 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
Coulter counter 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
Histograms RBCs, WBCs, plts
X-axis
- Horizontal/abscissa
- Size of cells
Y-axis
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- Vertical/ordinate
- Number of cells
V=IxR
Where:
V = voltage
I = current
R = resistance
Positive error
Bubbles
Extraneous electrical pulses
Aperture plug
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, Normochromic 1. Defective formation of RBCs or the presence of tumor cells in BM:
RBCs *Aplastic anemia
*Leukemia
*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
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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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