Routine
• Na2EDTA • K2EDTA • K3EDTA
3 Salts of EDTA
ARC = RBC count (x10^12) x Retic Count (%)/ 100
ARC formula
• Carboxyhemoglobin • Methemoglobin • Sulfhemoglobin
Abnormal Hemoglobin Pigments
Reagent: 0.1 N HCl Result: Brownish-yellow color solution Reading: Compared to standard comparator block
Acid Hematin/ Sahli's Method (Reagent, Result, Reading)
Eosin
Acid dye of Romanowksy stains
Excessive EDTA does not affect hemoglobin
Advantage of EDTA
Preserves V and VIII
Advantage of Na citrate
Even distribution of WBCs
Advantage of cover glass smears
1. Less volume of blood used 2. Less time 3. Better packing of cells
Advantages in Microhematocrit
200x more than oxygen
Affinity of hemoglobin to carbon monoxide
Lymphocytes and Monocytes
Agranulocytes
Reagent: 0.1N NaOH Reading: Compared to standard comparator block
Alkali Hematin (Reagent,Reading)
1. Forearm (ventral) 2. Wrist 3. Posterior part of hand 4. Foot, ankle
Alternate sites of venipuncture
• Trapped plasma • 1-3%
Amount of plasma that remained in RBC portion after spinning Results in manual method is _____% higher than results computed by automation
38%
An MCHC above ______% should not occur
< 15 degrees
Angle between syringe and patient's arm
30-40 degrees
Angle of Wedge smear
small, round, oval or elongated particles, highly refractive, light bluish color
Appearance of platelets under light microscope
9 mm^2
Area counted in Eosinophil count
1mm2
Area counted in platelet count
Methylene blue
Basic dye of Romanowksy stains
• Acute infections • Stress • Increased levels of glucocorticoids
Basopenia
1. Immune reactionsL • Immediate hypersensitivity reactions • Long -term foreign antigen stimulation 2. Certain conditions • Hypothyroidism • Ulcerative colitis • Myoproliferative disorders 3. Physiologic • Exercise • Estrogen therapy • Transient basophilia in newborn infants
Basophilia
dark blue black granules
Basophils in Wright Giemsa stain
Dacie's
Best diluting fluid
• Na citrate • Plasma: ESR-hematology • Chelates (binds) calcium
Black
_____%
Buffy coat is approximately _____ % of spun blood
• WBC count • RBC count • Hgb • Hct • Differenital Count • Platelet Estimate • Indices
CBC workup
CRC = Patient's Hct/45% x Retic count (%)
CRC formula
Massive/Excessive blood transfusion
Causes of Hypervolemia
• Loss of whole blood • Burns • Dehydration
Causes of Hypovolemia
1. Post-transfusion (normal RBCs transfused will show difference in size) 2. Post-treatment for Macrocytic Anemia (Iron, Vit B12 therapy) 3. Idiopathic Sideroblastic Anemia 4. Presence of two concurrent deficiencies (Iron and Folic Acid Deficiencies)
Causes of high RDW
1. Too large or too small drop of blood 2. Improper, jerky manner of spreading 3. Improper angle 4. Failure to keep entire edge of spreader slider against slide 5. Failure to push spreader slide completely across slide
Causes of poor PBS
• Cell Count = # cells x Dilution Factor x Volume Correction Factor • VCF = 1/TV • TV = # squares x dimensions ( LxWxDepth) Shortcut (for routine only): • RBC = # cells x 10,000 • WBC = # cells x 50
Cell Count Formulas
2,000 to 2,300 x g for 30 minutes
Centrifugation of Macrohematocrit Method
• Fingertips: 3rd or 4th finger • Earlobe • Plantar surface of foot: medial or lateral for newborn
Collection sites in capillary Blood
1. Acid Hematin (Sahli's Method) 2. Alkali Hematin 3. Direct Matching
Colorimetric Methods
• Phloxine - stain • Na2CO3 - lyses other WBCs and enhances staining • Propylene glycol - lyses RBCs
Components of Phloxine diluting fluid
Phloxine diluting fluid + 100 units of Heparin
Components of Pilot's fluid
Solution 1: Methylene Blue + water Solution 2: Phloxine + water
Components of Randolph's stain
• Venous Blood • Arterial Blood • Tissue Fluid
Composition of Capillary Blood
• Protoporphyrin ring • Fe 2+
Composition of Heme
Macromolecules: • CHON • CHO • Lipids Micromolecules (ions): • salts • hormones • nitrogenous substances • trace elements, and vitamins
Composition of Liquid Portion
• Absolute Retic Count • Corrected Retic Count • Reticulocyte Production Index
Computations on Reticulocyte Count
80-100% HbS
Concentration of HbS in Sickle Cell anemia
20-40% HbS
Concentration of HbS in Sickle Cell trait
• 34% of normal RBC • 85% of dry weight of RBC • MW = 64,000 Da
Concentration of Hemoglobin in Blood
1-10% concentration in blood
Concentration of carboxyhemoglobin in blood of tobacco smokers
1. Myeloma 2. Abnormal Globulins 3. Radiographic Contrast Method
Conditions that Affect Copper Sulfate Method
• Splenectomy • Liver Disease • Sickle Cell • IDA • Thalassemia > > target cells • Any conditions where target cells are found
Conditions with Decreased OFT
•Hereditary Spherocytosis • Hemolytic Anemia • Any conditions wherein spherocytes are found
Conditions with Increased OFT
• Pancytopenia • Aplastic Anemia • Sideroblastic Anemia • Thalassemia
Conditions with Increased Retic Count
Reticulocytes tend to settle on top of mature RBCs --> mix blood and stain well
Consideration in Retic Count
• less free nerve endings --> less pain • less admixture or tissue juice • for newborns but not NBS • performed in Duke's Method (BT)
Considerations in Earlobe Collection
• Use opposite arm • If not possible, stop IV for 2 minutes and extract blood below IV line • Not applicable to glucose and phosphorus
Considerations in collecting from patients with IV
• EDTA decreases clumping of platelets but causes an increase in the MPV during the first hour in tube • Lower in capillary blood
Considerations in platelet count
Turbidity or clouding of solution will falsely increase the hemoglobin value 1. Excessive increase in WBC ---> centrifuge and use supernatant 2. HgbS and Hgb C ---> resistant to lysis; dilute specimen 1:1 with dist. H2O, result x2 3. Lipemia ---> use patient blank * increased Protein has no effect due to nonionic detergents
Considerations of Cyanmethemoglobin and Remedies
1. Posture 2. Diurnal Rhythm 3. Exercise 4. Stress 5. Diet 6. Smoking
Considerations that Affect Test Results
PInk top
Contains label prescribed by AABB
HbF - resistant to alkali substances - however, NaOH is used in Kleihauer-Betke and APT test
Contraindicated in Alkali hematin
• Cushing's disease • ACTH administration (used in conjunction with the Thorn test for adrenal cortical function) • shock
Decreased Eosinophilia
Microcytic, hypochromic RBCs (except spherocytes)
Decreased MCH
Hypochromic: • IDA • Thalassemia
Decreased MCHC
Microcytic: • IDA • Thalassemia • Sideroblastic Anemia • Lead poisoning
Decreased MCV
* Diluted by tissue fluid • Hemoglobin • Hematocrit • RBC Count • Platelet count - adheres to site of injury
Decreased parameters in capillary blood
0.1 mm
Depth of Neubauer counting chamber
1. Glucose, Sodium - increased 2. CBC - increased *Hgb after eating is increased due to turbidity; measured via spectrophotometry (Cyanmethemoglobin)
Diet
0.5% saline
Diluent used in Sanford Method
•Phloxine diluting Fluid • Pilot's solution • Randolph's stain
Diluting fluids in Eosinophil Count
1:100 or 1:200 dilution
Dilution of Platelets
1:32
Dilution used in Phloxine
If WBC count is: • <3 x 10^ 9/L, use 1:10 or 1:11 • > 30 x 10 ^ 9/L, use 1:100 or 1:101 • 100-300 x 10^ 9/L, use 1:200 or 1:201
Dilutions of WBC Count
• Tallqvist • Dare • Spencer
Direct Matching Methods
1. Microhematocrit 2.Macrohematocrit Method (obsolete)
Direct Methods of Hematocrit
• Causes prolonged coagulation tests • prone to increased anticoagulation in PCV patients
Disadvantage of 3.8% Na Citrate
Cannot distinguish sickle cell trait vs sickle cell anemia
Disadvantage of Na metabisulfite method
• Time consuming • Difficult to master • For most automated strainers, cover glasses are too small, hard to label and easily broken
Disadvantage of cover glass smears
cannot be used for PBS because it distorts cells and causes blue background
Disadvantage of heparin
• V is not stable • inhibits thromin-fibrinogen reaction • Platelet satellitism - platelets adhere to PMNs --> yields false decreased automated platelet count
Disadvantages of EDTA
1. Time-consuming 2. Larger amounts of blood needed 3. Higher amounts of trapped plasma present
Disadvantages of Macrohematocrit Method
• Thixotropic: changes viscosity upon centrifugation • Cannot be used for TDM as it can absorb some drugs
Disadvantages of separation gel
1. ACTH, Cortisol - increased (8am) 2. Iron, Eosinophils - increased in the afternoon (Henry - iron increased in morning) 3. Neutrophil - increased in the afternoon
Diurnal Rhythm
1%
Duplicate results should agree within ____%
• Methylene blue • Eosin B or Y
Dyes in Romanowsky stains
• Decreased production • Acute Bacterial infection • ACTH administration
Eosinopenia
bright orange granules
Eosinophil in Wright Giemsa stain
1. Infections • Infestation by tissue-invading parasites • Scarlet fever 2. Allergic reactions • Respiratory- asthma, hay fever • Skin disorders - psoriasis, eczema 3. Malignancies of myeloid cells 4. Hypersensitivity disorders • Loeffler's syndrome • Pulmonary infiltrates with eosinophilia • Tropical Eosinophilia 5. Miscellaneous • Familial • Posterradiation • Periarteritis nodosa • Certain poisons
Eosinophilia
• Microhematocrit tube - 50 uL - 7-7.5 cm x 1 mm - Filled to at least 5 cm or 3/4 of tube • Microcentrifuge: 10,000 - 15,000 x g for 5 mins • Clay: 4-6 mm
Equipment and Measurements in Microhematocrit
• Thick films • Prolonged staining time • Inadequate washing • Too alkaline stain or diluent • Short drying period
Excessively blue staining
• Insufficient staining • Prolonged washing time • Mounting the cover slips before they are dry • Too acidic stain or buffer • Old stain (methanol --> formic acid)
Excessively pink staining
1. Creatinine, Protein, CK, AST, LD - increased 2. Coagulation, Fibrinolysis - activated 3. Platelet and WBC count - increased
Exercise
0.02
Factor in Sanford Method
• Plasma concentration • RBCs • Mechanical or Technical Factors
Factors affecting ESR
1. Incompletely sealed tubes 2. Overanticoagulation
False decreased microhct
1. Prolonged standing of tube before reading --> pRBC will loosen 2. Undercentrifugation
False increased microhct
•Overanticoagulation • Reading before 60 minutes • Decreased temperature
Falsely decreased ESR
• Reading after 60 minutes • Increased temperature • Tilting of tube
Falsely increased ESR
Methanol
Fixative of Romanowksy stains
WBC count x 100/ # NRBC + 100
Formula for Corrected WBC Count
Retic% = #retics x 100/ # RBCs x 9
Formula of Miller Disk
average of platelet count in 10 fields x 20,000
Formula of Platelet estimation
Retic count (%) = #Retics/10
Formula of Retic Count
Amt. of citrate = (100-Hct)/ (595-Hct) x whole blood (mL)
Formula of amount of citrate
Copper Sulfate Method/ Gravimetric Method
Frequently used in blood donor screening If hemoglobin level is acceptable, drop of blood will sink within 15s. Should be added from a height of approx. 1cm
binds Mg2+ which is needed by Enolase in glycolytic pathway
Function of Na fluoride in inhibiting glycolysis
Formed Elements (45%) Liquid Portion (55%)
General Components of Blood
B,E,N
Granulocytes
• Na fluoride (anti-glycolytic) / K oxalate (anticoagulant) • Plasma: glucose testing • Inhibits glycolysis, chelates calcium
Gray
• Na heparin/ Lithium heparin • Plasma: chemistry • Inhibits thrombin formation; enhances Antithrombin III
Green
• 40-45% = 1 • 35-39% = 1.5 • 25-34% = 2 • 15-24% = 2.5 • <15 = 3
Hct Value and corresponding Maturation Time (correction factor)
• Cellulose acetate • Citrate Agar
Hemoglobin Electrophoresis media
= 0.34g Fe/ 100g Hgb
Hemoglobin iron content
"Huffner's Factor" 1.34 mL O2 / 1g Hgb
Hemoglobin oxygen capacity
100 cells
How many cells are counted on routine differential count?
23g - blue/torqoise 22g - black 21g - green 20g- yellow 18g - pink
Hub colors of: 23g 22g 21g 20g 18g
1+ = area of central pallor is 1/2 of cell diameter 2+ = area of pallor is 2/3 of cell diameter 3+ = area of pallor is 3/4 4+ = thin rim of hemoglobin
Hypochromia Grading
1.1
If specimen is obtained using 0.109M Na citrate, multiply results by ______
• Unclean slides • Inadequate washing of the slide at the end of the staining period • Failure to hold the slide horizontally during the initial washing • Inadequate filtration of stain • Permitting dust to settle on the slide or smear
If there are precipitates on the film, these might be due to:
Lymphocytes
Immunocytes
Absolute Retic Count
In 1L of whole blood, this measures the actual number of reticulocytes
30-60 minutes
In red top, blood must clot within _________ before centrifuging, otherwise RBCs will lyse
• Allergic Reactions • Parasitic Infections • Brucellosis • Certain Leukemias
Increased Eosinophilia
Macrocytic anemias (larger RBCs carry more Hgb)
Increased MCH
Hyperchromic: • possible error in RBC or hemoglobin measurements • presence of spherocytes
Increased MCHC
Macrocytic: • Megaloblastic Anemia • Hemolytic Anemia with reticulocytosis • Liver disease • Hypothyroidism • Normal Newborns
Increased MCV
• Chronic and Acute blood loss • Hemolytic Anemia • IDA with iron therapy
Increased Retic Count
WBC count due to injury
Increased parameter in capillary blood
Stratified sedimentation
Increased reticulocytes tend to float, RBCs and plasma are poorly separated
RPI >3
Indicates adequate BM response
RPI <2
Indicates inadequate BM response
1. Newborn 2. Pediatric patients: < 2 years old 3. Severely burned patients (veins are reserved for therapeutic purposes) 4. Geriatric veins (fragile veins)
Indications of Capillary Puncture
Markedly decreased: 0-49,000/uL Moderately decreased: 50,000-99,000/uL Slightly decreased: 100,000-149,000/uL Low normal: 150,000-199,000/uL Normal: 200,000-400,000/uL Slightly increased: 401,000-599,000/uL Moderately increased: 600,000-800,000/uL Markedly increased: Greater than 800,000/uL
Interpretation of Platelet estimates
• Bubbles • Fibrin clots
Invalid results in ESR
• K3EDTA • Whole blood: Hematology • Chelates (binds) calcium
Lavender (Glass)
• K2EDTA/ Spray-dried • Whole blood: Hematology • Chelates (binds) calcium
Lavender (Plastic)
1. Fat 2. Plasma 3. Buffy coat 4. Packed RBC
Layers of Spun Microhematocrit
• Na citrate • Plasma: coagulation • Chelates (binds calcium)
Light Blue
• Thrombin and soybean trypsin • Plasma: coagulation • Fibrin degradation products
Light Blue (2)
• Lithium heparin and gel • Plasma: chemistry • Inhibits thrombin formation; enhances Antithrombin III
Light green/black
dark purple nuclei varying shades of blue cytoplasm
Lymphocytes in Wright Giemsa stain
• Long-term drug therapy • Immunodeficiency
Lymphocytopenia
• Viral infections: EBV • Whooping cough • Lymphocytic leukemia
Lymphocytosis
• NV: 26-34 pg • Indicates the average weight of hemoglobin in individual RBCs • Less valuable to the clinician than other indices • Formula: MCH = Hb (g/dL)/ RBC count x 10
MCH
• NV: 32-37 g/dL • Indicates the average concentration of hemoglobin • MCHC = Hb (g/dL)/ Hct x 100
MCHC
• 22% • presence of Hb S and Hb C
MCHC will not fall below __________% when hypochromia is present. If it does, it may be due to ____________
Antecubital fossa
Main site of venipuncture
Abs. Eo Count = WBC ct x % Eo (Relative count)
Manual Absolute Eo Count formula
Direct Matching
Matching of the red color of fresh blood with different color standards
• NV: 80-100 fL • Indicates the average volume of RBC • Formula: MCV = Hct/RBC x 10
Mean Cell Volume (NV, Definition, Formula)
115 mm long 3 mm internal bore * for Macrohct: 100 at the top (right calibration) * for ESR: 0 at the top (left calibration)
Measurements of Wintrobe
Osmotic Fragility Test
Measures ability of RBCs to take up fluid without being lysed
• A tilt of 3 degrees can cause errors up to 30% • Vibration alters results • Narrower bores decreases ESR
Mechanical or Technical factors
Westergren method
Method that is more sensitive in patients with high ESRs
Wintrobe Method
Method that is more sensitive in patients with low ESRs
1. Blood Oxygen Capacity/ Gasometric/ Van Slyke Method 2. Blood Iron Content 4. Copper Sulfate Method/ Gravimetric Method 5. Colorimetric (Acid Hematin/Sahli's Method, Alkali Hematin, Direct Matching) 5. Oxyhemoglobin Method 6. Cyanmethemoglobin/ Hemiglobincyanide
Methods in Determining Hemoglobin Levels
• Sodium metabisulfite method • Sodium dithionate method (Solubility test) • Hemocard HbA and HbS
Methods in Sickle Cell Examination
1. Crenellation/Cross-sectional 2. Longitudinal method 3. Battlement method
Methods of Differential Count
• Westergren • Wintrobe
Methods of ESR
• Indirect: Diff. Count • Direct: Eosinophil Count
Methods of Eosinophil Count
1. Microscopy 2. Automated cell counters 3. Estimation using smears
Methods of Platelet Count
1. Phase Contrast Microscopy 2. Toncantin Method (Reese&Ecker)
Microscopic Methods of Platelet Count
(-) C,E,O,A2 < S,D,G< A1 (Middle) < Bart's < I < H (+)
Migration of Hemoglobin in Cellulose Acetate
(-) F< A,E < ORIGIN < D,O,G < S < C (+)
Migration of Hemoglobin in Citrate Agar
112 cells *112 cells = 1008 cells in A
Mininum number of cells counted in square B
lighter purple nucleus gray blue cytoplasm
Monocyte in Wright Giemsa stain
• Overwhelming infections causing neutropenia • Inherited • Increased levels of glucocorticoids
Monocytopenia
1. Bacterial infection • Tuberculosis • Subacute bacterial endocarditis • Syphilis 2. Recovery from neutropenia (relative) 3. Inflammatory response • Surgical trauma • Tumors • Collagen vascular diseases • Gastrointestinal disease 4. Myoproliferative disorders
Monocytosis
Monocytes and Lymphocytes
Mononucleated
Evacuated tube system
Most common method of venipuncture
• Routine: 1 inch • Very deep veins: 1.5 inches
Most common needle lengths
21g
Most common needle size
70% Isopropyl Alcohol
Most common skin cleanser
25-75 x 10^ 9/L
NV of Absolute Retic Count
Males: 0-10mm/hr Females: 0-15mm/hr
NV of Westergren ESR
Males: 0-9 mm/hr Females: 0-20 mm/hr
NV or ESRs in Wintrobe method
Decreased Neutrophil Production: 1. Inherited stem cell disorders 2. Acquired stem cell disorders: • Chemical toxicity (benzene) • Cytotoxic drugs • Marrow replacement • Nutritional deficiencies Increased Neutrophil destruction: • Infections (Overwhelming bacterial/viral infections • Immune reactions • Sequestration • Pseudoneutropenia • Malignant myeloproliferative disorders
Neutropenia
1. Infections: • Bacterial infection (pyogenic bacteria, spirochete and rickettsia, certain viruses, actinomyces fungi, N. fowleri 2. Inflammatory response • Neoplastic growth • Metabolic disorders • Acute hemorrhage 3. Physiologic • Response to therapy (Corticosteroids, Lithium) • Pseudoneutrophilia (Stress, Physical or emotional stimuli)
Neutrophilia
dark purple nuclei light pink cytoplasm with lilac granules
Neutrophils in Wright Giemsa stain
50-350 x 10^6/L
Normal Eosinophil Count
• Initial hemolysis at tube 22 • Complete hemolysis at tube 17
Normal Value of OFT
• Neutrophil: 35-71% • Band: 0-6% • Lymphocyte: 24-44% • Monocyte: 1-10% • Eosinophil: 0-4% • Basophil: 0-2%
Normal Values for Differential Count
• Birth: 10-30 x 10 ^ 9/L • 1 Year: 6-17 x 10 ^ 9/L • Normal Adult: 4-11 x 10 ^9/L
Normal Values in WBC Count
• Birth: 5-6.5 x10 ^ 12/L • 1 year: 3.5-5.1 x 10^ 12/L • Adult Female: 3.6 - 5.6 x 10 ^ 12/L • Adult Male: 4.2-6.0 x 10 ^ 12/L
Normal Values of Red Blood Cell Count
1-5%
Normal individuals have up to ____% methemoglobin
8-20 platelets/OIF
Normal platelet count per field
SG 1.053 = 12.5 g/dL hemoglobin SG 1.055 = 13.5 g/dL
Normal value of Copper Sulfate Method
150,000-450,000/uL
Normal value of Platelet count
11.5-14.5%
Normal value of RDW
Adult: 0.5-1.5% Newborn: 2-6%
Normal value of Retic Count
• Birth: 45-60% • 1 year: 27-44% • Adult Female: 36-48% • Adult Male: 40-55%
Normal values of Hematocrit (Packed RBC)
Birth: 15-20 g/dL 2 months: 9-14 g/dL 10 years: 12-15 g/dL Adult females: 12-16 g/dL Adult males: 13-18 g/dL * Hgb F has more affinity to O2 * Females: monthly blood loss and less testosterone
Normal values of Hemoglobin
3-4 drops of blood
Number of drops needed in Automated Spun smear or Automated hemaspinner
10x
Objective used in Eosinophil count
40x
Objective used in Platelet count (Phase contrast)
40x
Objective used in RBC count
10x
Objective used in WBC count
1. Blood gases 2. Slides 3. EDTA 4. Other anticoagulants 5. Serum
Order of Draw (Skin puncture)
1. Blood culture 2. Citrate 3. Non-additive 4. Heparin 5. EDTA 6. Fluoride
Order of Draw of ETS
Used NaHCO3 isntead of H2KPO4
Original Drabkin's
1. Count only 50 cells when WBC count is <1x10^9/L; Alternatively, a buffy coat smear, which has concentrated WBCs may be prepared .2 Count 200 cells if: • Over 10% eosinophils • Over 2% Basophils • Over 11% monocytes • More lymphocytes than neutrophils except in children Afterwards, average the results (divided by 2)
Other considerations in Diff count
• C (Harlem) • S (Travis) • C (Ziguichor)
Other sickling Hb
• vacuolization of granulocytes • distortion of lymphocytic and monocytic morphologies • crenation of RBCs
Oxalate is not used in cell morphology analysis because it causes:
sulfhemoglobin and methemoglobin
Oxyhemoglobin method does not measure:
Cover glass smears (Ehrlich)
PBS for BM preparation
Thick blood smear
PBS used in malaria
• High blood sugar • Use of heparin
Pale staining in Retic Counts are caused by:
7%=8%
Percent of blood in body weight
within 2 hours
Peripheral Blood Smear is done within:
• Basophiles • Eosinophils • Neutrophils • Monocytes
Phagocytes
• Spray-dried K2EDTA • Whole blood: BLOOD BANK AND MOLECULAR DIAGNOSTICS • Chelates (binds) calcium
Pink
violet to purple
Platelet in Wright Giemsa stain
Romanowsky stain
Polychromatic stain
B,E,N
Polynucleated
Increased Lipids and Proteins: - Prolonged standing: increased hydrostatic pressure - Lying Down: decreased hydrostatic pressure - increased bilirubin (adheres to albumin) * Micromolecules accompany fluid; Macromolecules (lipids, proteins) remain in blood vessel
Posture
3.2%
Preferred concentration of Light blue top
Hgb (Fe2+) ---KFCN---> Methgb (Fe3+) ---KCN----> Cyanmethgb (540nm)
Principle of Cyanmethemoglobin
• During tissue injury, acute phase response occurs • Plasma concentration of APRs increase • These APRs destroy the RBCs' shield of negativity and affects ESR
Principle of ESR
• Whole blood is diluted with isotonic diluting fluid to prevent lysis of RBCs • In PCV and severe dehydration, RBC count increases • In cases of anemia, aging and after meals, RBC count decreases
Principle of Manual RBC Count
While blood is diluted with weak acid leading to the lysis of RBCs
Principle of Manual WBC Count
• Saponin lyses RBCs, releasing hemoglobin • HbS and other sickling Hb in the reduced state forms liquid crystals and yield (+) turbid appearance
Principle of Na dithionate method
Na metabisulfite deoxygenates Hb --> (+) HbS present to sickle; Holly leaf form
Principle of Na metabisulfite method
• 1% Ammonium oxalate hemolyzes RBCs • Platelets are counted using the phase contrast microscope
Principle of Phase contrast microscopy
• Any non-nucleated RBC with two or more particles of blue-stained, granulofilamentous material after NMB staining is defined as a reticulocyte • The number of reticulocytes in 1000 RBCs is determined and divided by 10 to get RC in %
Principle of Reticulocyte Count
The degree of sickling depends on HbS concentration in RBC
Principle of Sickle Cell Eamination
Unlike reference method, RBCs are not hemolyzed and uses light microscope
Principle of Toncantin Method
Albumin and Lecithin * Increases viscosity of blood
Proteins that decrease ESR
should not exceed 2 mm due to risk of bone injury and possible infection (Osteomyelitis, S. aureus)
Puncture depth of Skin puncture and considerations
• Gower • Hayem • Toisson • Bethell • Strong • Eagle • NSS (0.85%) • Dacie's
RBC Diluting fluids
• Larger, heavier, denser RBCs fall faster • Agglutination, rouleaux, macrocytes, severe anemia, adults >60 yo (decreased albumin) ---> Increased rate • Anisocytosis, poikilocytosis (Sickle cells, spherocytes), severe anemia, PCV --> decreased rate
RBC Factors in ESR
• Neubauer Counting chamber - most common; 2 chambers • Fuchs-Rosenthal • Speirs-Levy
RBC Hemocytometers
• RBC Unopette/Thoma RBC pipette • 20 ul pipet
RBC Pipet Description
• Surface Area • Volume • Functional state of RBC membran
RBC shape depends on:
pink to orange
RBCS in Wright Giemsa stain
• RBC histogram • Coefficient of variation of MCV
RDW is determined from:
RPI= CRC/maturation time
RPI formula
9:1
Ratio of blood to anticoagulant in Light Blue top
4:1
Ratio of blood to anticoagulant in black top
• Spectrophotometrically at 540nm • All forms of hemoglobin can be measured through this method except sulfhemoglobin
Reading of Cyanmethemoglobin Method
Modified Drabkin's Reagent: • K Ferricyanide (KFCN) • Nonionic Detergent - lyses RBC to release hemoglobin and decreases turbidity due to CHON precipitates • Dihydrogen Potassium Phosphate (H2KPO4) - allows reading after 3 mins instead of 15 mins
Reagent in Cyanmethemoglobin and Uses
Reese and Ecker Diluent: • Brilliant Cresyl Blue • Sodium Citrate • Formaldehyde • Distilled water
Reagent of Toncantin Method
• Pipets • Diluting Fluids • Hemocytometers
Reagents and Materials in WBC Count
• Pipets • Diluting Fluids • Hemocytometers
Reagents and Materials used in RBC Count
• Incorrect calculation of MCHC • Patient's RBCs may be agglutinated (cold agglutinins), thereby causing falsely low RBC count
Reason why MCHC must not be above 38%
They cause transfer of cells from marginating pool to circulating pool
Reason why elevated Cortisol and Epinephrine cause elevated WBC count
• shield of negativity • bound to haptoglobin • toxic to kidneys
Reasons why Hemoglobin is filtered by glomerulus
• None (negatively charged surface) • Serum: Chemistry and Serology • N/A
Red (Glass) • Anticoagulant/Additive • Specimen and Use • Mechanism of Action
• Clot Activator • Serum: Chem and Sero • Silica clot activator
Red (Plastic/Hemogard)
• Mean Cell Volume • Mean Cell Hemoglobin • Mean Cell Hemoglobin Concentration
Red Blood Cell Indices
• Clot activator and separation gel • Serum: Chemistry • Silica clot activator
Red/Gray and Gold
Phase Contrast Microscopy
Reference method of platelet count
Reticulocyte Count
Reflects the amount of effective RBC production in the bone marrow
• Directly proportional • As the degree of anisocytosis increases, the RDW also increases
Relationship of RDW and anisocytosis
Falsely elevated due to fewer mature RBCs (more RBCs are being counted), therefore CRC is used
Result of Retic count with low Hct
decreased
Retic count in Anemia caused by decreased production
increased
Retic count in Anemia cuased by increased destruction
• stays in BM 2-3 days • only stage found in both BM and PBS • last immature RBC stage
Reticulocytes
pinkish gray
Reticulocytes in Wright Giemsa stain
• Wright's • Giemsa • Modified Wright's-Giemsa (blood parasites) • Leishman • Jenner • May Grunwald
Romanowksy stains
1:20
Routine WBC Dilution
1:10
Routine dilution of Eosinophil Count
1:200
Routing dilution of RBC
• Na heparin, Na2EDTA • Plasma: Chemistry/ Toxicology • Heparin inhibits thrombin formation; Na2EDTA binds calcium
Royal Blue
RBC x 3 = Hemoglobin Hgb x 3 = Hct +/- 3 (%)
Rule of 3
Nonspecific test to detect and monitor inflammation
Significance of EST
• Howell-Jolly bodies • Heinz bodies • Hb H • Pappenheimer bodies
Since supravital stain is used in Retic count, other inclusions may be stained such as:
1. WBC count - increased 2. Cortisol - increased 3. Hemoglobin, Hct, RBC - increased * Smoking is a form of stress * dysfunctional lungs cause impaired delivery of O2, stimulating kidneys to produce EPO * Smokers are difficult to collect in skin puncture due to impaired circulation
Smoking
Reduce amount of citrate
Solution to overanticoagulation caused by Na citrate
• EDTA or Heparinized Whole Blood • Capillary Blood
Specimen for RBC Count
EDTA whole blood or Capillary blood
Specimen for Retic Count
EDTA Whole Blood or Capillary Blood
Specimen in Cyanmethemoglobin method
• K2EDTA • K3EDTA causes 2-3% decrease in hematocit due to slight shrinkage of cells
Specimen in Microhematocrit
Heparinized blood - maintains blood at pH 7.4 which is required for the test
Specimen in OFT
• EDTA whole blood • Capillary Blood
Specimen used in WBC Count
A
Square where retics are counted in Miller Disc
Large central square
Squares counted in RBC Count
4 large corner squares
Squares counted in WBC Count
• Lag phase: 10 minutes (little sedimentation --> Stage of rouleaux) • Decantation phase: 40 minuttes (settling occurs at constant rate-->Stage of Sedimentation) • Slow sedimentation : 10 minutes (due to accumulation of RBCs in the bottom)
Stages of Erythrocyte sedimentation
1. WBC count - increased 2. Acid-base imbalance - hyperventilation - respiratory alkalosis 3. Cortisol and Epinephrine - increased
Stress
4 Heme 4 Globin chains
Structure of Hemoglobin
Reticulocyte Production Index
Takes into consideration the maturation time of RBCs
• Na heparin • Plasma: Lead Testing • Inhibits thrombin formation
Tan (Glass)
• K2EDTA • Plasma: Lead Testing • Chelates (binds) calcium
Tan (Plastic)
• CBC • Counts: Platelets, Reticulocyte, Eosinophil • Hb electrophoresis • Sickle cell preparation • PBS
Tests performed on EDTA
• OFT and Platelet Retention Test • Barbiturates • BUN • Carboxyhemoglobin • Cholinesterase • Cortisol • Fibrinogen • Methemoglobin • Ammonia • Na,K,Cl, CO2, pH • Sulfa Level • Haptoglobin • Plasma Hemoglobin • Copper
Tests performed on Heparin
• Blood Alcohol • Glucose Tolerance • Lactic acid and Tolerance • Pyruvic acid
Tests performed on Heparin + NaF (gray)
Blood gases
Tests performed on Heparinized syringe
• Coagulation studies • Platelet Aggregation • Standard Westergren
Tests performed on Na Citrate
15%
There is approximately ______% error for manual WBC Count
Decreased: (P)ressure Increased: (A)ngle (S)ize (S)peed
Thick smear
Increased: (P)ressure Decreased: (A)ngle (S)ize (S)peed
Thin smear
•Polycythemia vera •CML •Splenectomy (30% platelets are sequestered by spleen) •Idiopathic thrombocytopenia
Thrombocythemia
• Thrombocytopenic purpura • Aplastic Anemia • Acute Leukemia • Gaucher's disease • Pernicious Anemia • Chemotherapy • Radiation therapy • Splenomegaly
Thrombocytopenia
Isotonic
Tonicity of RBC diluting fluid
Blood Iron Content
Total iron content of blood may be regarded as being bound to hemoglobin
5-6 L
Total volume of blood
• 3-4 inches (Rodak: 2-4 inches)/ 7.5-10cm from site • Duration: <1 minute • >1 minute can produce hemoconcentration due to increased hydrostatic pressure
Tourniquet considerations
1. Macrocytic anemia 2. Thalassemia 3. Spherocytosis 4. Hypochromic Anemia 5. Sickle Cell anemia
Trapped plasma is increased in:
Hyperbaric Chamber to displace carbon monoxide
Treatment for Carboxyhemoglobin
1. Cover glass smears (Ehrlich) 2. Wedge smear 3. Automated Spun smear (Automated hemaspinenr) 4. Buffy coat smear 5. Thick blood smear
Types of PBS
Heparin
Universal anticoagulant
• Highest during the night (midnight and later) • Lowest in late morning
Variation in Eosinophil count
• Morning - increased • Evening - decreased • Strenuous muscular activity - increased • Smokers - increased • Increasing altitude - increased • Aging - decreased * high altitudes = low pO2 --> hypoxia
Variation of concentration of Hemoglobin
1. Strenuous physical activity - increased 2. Dehydration and altitude - increased ( decreased plasma) 3. Highest in the morning, lowest in the evening
Variations in RBC Count
1. Slightly higher in the afternoon than in the morning 2. Greater variation in disease among children (extreme increase or decrease) 3. Strenuous exercise, emotional stress, and anxiety will increase counts
Variations in WBC Count
• 1-3% Acetic Acid • 1% Hydrochloric Acid • Turk's Diluting Fluid
WBC Diluting Fluids
Neubauer counting chamber
WBC Hemocytometer
• WBC Unopette • Thoma White cell pipet • 20 uL pipet
WBC Pipet description
• Granules • # of Nucleus • Function
WBCs are classified according to:
results to specimen resembling arterial blood
Warming of puncture site
630nm
Wavelength Methemoglobin is read
618 nm
Wavelength at which Sulfhemoglobin is read
576 nm
Wavelength used in Carboxyhemoglobin
• 300 mm long • 2.55 mm internal bore • Calibrated from 0-200
Westergren measurements and calibration
• cells • 1/9 of A
What is counted in square B? What is the size of square B?
• => 5 nRBCs in adult • => 10 nRBCs in newborn
When is corrected WBC count performed?
• EDTA and gel • Plasma: molecular diagnostics • Chelates (binds) calcium
White
It is prone to infection leading to gangrene
Why is foot/ankle avoided in patients with DM or hemoglobinopathies
• 115 mm long • 3mm internal bore • Calibration: 0 at the top (left calibration)
Wintrobe measurements and calibration
• Sterile containing SPS • Serum: Microbiology culture • Aids in bacterial recovery by inhibiting: complement, phagocytosis and antibiotics
Yellow
• Acid Citrate Dextrose • Plasma: BB, HLA, Phenotyping, Paternity Testing • WBC preservative
Yellow (2)
• Thrombin • Serum: Chemistry • Clot Activator
Yellow/Gray and Orange
5 minutes
Yellow/Gray and Orange (Thrombin) tops must clot within _______ before centrifugation
Basophils
affinity for alkaline dye; purple-black granules of unequal size; scattered over nucleus and cytoplasm
Eosinophils
affinity for eosin or red dye; bright red granules of equal size
Na2EDTA
aka "Versene"
Hb Bart
also positive for Na dithionate
Iodoacetate
alternative for Na Fluoride
Nucleated RBCs
are not lysed by hypotonic solutions, leading to falsely elevated WBC count
Battlement method
begin counting near the tail on a horizontal edfe: count three consecutive horizontal edfe fields, count two fields toward the center, count two fields horizontally, count two fields vertically to the edge
Brilliant Cresyl Blue
causes too much inconsistency, therefore NMB is used
Crenellation/Cross-sectional
counting of WBCs in consecutive fields as the blood film is moved from side to side
2-3 mm
diameter of drop of blood
0.25 in
distrance of blood drop from edge of slide
Longitudinal method
from tail toward the head of the smear
Shift to the right
increased number of hypersegmented neutrophils. Absolute count is more preferred
Liquid Portion
made up of 90% water and 10% glucose, proteins, carbohydrates, lipids, and salts. Hormones, nitrogenous substances, ions, trace elements, and vitamins are also found in the liquid portion. a. Plasma b. Serum
Infectious crisis
most common cause of death of patients with sickle cell
Phloxine diluting fluid
most common diluting fluid in Eosinophil Count
tube 14
most hypotonic tube
Azure B
oxidation product of methylene blue
• Alkaline • pH 8.6
pH of Cellulose acetate
• Acid • pH 6.0 - 6.3
pH of Citrate Agar
7.4
pH required in OFT
Neutrophils
pinkish-lavender granules of unequal size
K2EDTA
preferred over K3EDTA
Shift to the left
presence of immature granulocytes (Leukemia and bacterial infections)
Shape of RBCs
primary factor of OFT
6.8
recommended pH in BM staining
7.2
recommended pH in staining Schuffner's granules
zeta potential
referred to as the negative charge of RBCs
sialylated glycoprotein
responsible for the zeta potential of RBCs
Plasma concentration * As APRs increase, there is also an increase in the rate of rouleaux formation
single most important factor of ESR
• smallest surface area • increased OFT
surface area and OFT of Spherocytes
• largest surface area • decreased OFT
surface area and OFT of Target cells
Formed Elements
this portion is made up of cells or hemocytes: a. packed cell volume b. buffy coat
hypotonic
tonicity of tube 25
10% aqueous Chlorox
used in cleaning WBC Thoma pipets
Sanford Method
• 12 tubes labeled 14-25 • label of tube corresponds to number of drops of the diluent • add drops of dist. water until the total number of drops (diluent + dH2O) equates to 25 in all tubes
Blood Oxygen Capacity/ Gasometric/ Van Slyke Method
• Estimates oxygen capacity of blood • Measures functional hemoglobin only • relatively inaccurate (2-12% Hgb is inactive)
Methemoglobin
• Fe2+ oxidized to Fe3+ • Acquired (chemicals, drugs) or inherited • Chocolate brown discoloration of blood • Reversible • can cause cyanosis and functional anemia
Rule of Three
• For quality control only • Only applicable to normocytic and normochromic RBCs
Sulfhemoglobin
• Formed by action of certain drugs such as sulfonamides and aromatic amines • Mauve-lavender coloration of blood • Irreversible • can form carboxysulfhemoglobin • Not normally found in blood
Carboxyhemoglobin
• Hgb + Carbon monoxide • Brilliant, cherry-red color of the face • Reversible
Na dithionate method
• Most common method • Used for screening
Cyanmethemoglobin method
• Routine method • Done manually or by automated machines • Most reliable method
Katayama's Test
• Test for Carboxyhemoglobiin • uses ammonium sulfide • can detect as little as 10% carboxyhemoglobin concentration
Buffy coat smear
• Used in patient with WBC count <1x10^9/L • Concentrates the nucleated cells (if present) • LE cell preparation
Oxyhemoglobin
• Used to measure plasma hemoglobin • Not affected by moderate bilirubinemia
K3EDTA
• aka "sequestrene" • liquid • causes dilution and shrinkage of cells