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