INTRODUCTION TO CLINICAL HEMATOLOGY powerpoint

HEMATOLOGY
(INTRODUCTION)
• HEMATOLOGY (STUDY OF BLOOD CELLS)
• RED BLOOD CELLS/RBC = ERYTHROCYTES
• (RESPIRATION)
• WHITE BLOOD CELLS/WBC = LEUKOCYTES
• (IMMUNE FUNCTION)
• PLATELETS = THROMBOCYTES
• (HEMOSTASIS: CLOTTING AND BLEEDING)
• BLOOD = RESPIRATORY FLUID THAT CIRCULATES
ALL OVER THE BODY

HEMATOLOGY
(INTRODUCTION)
• COMPONENTS OF BLOOD
• 1. LIQUID (GLUCOSE, PROTEINS, LIPIDS, ETC.)
• SERUM (CLOTTED WITHOUT FIBRINOGEN)
• PLASMA (UNCLOTTED WITH FIBRINOGEN)
• 2. GAS (O2, CO2, CO)
• 3. SOLID (RBC’s, WBC’s, PLATELETS)

HEMATOLOGY
(INTRODUCTION)
• FUNCTIONS OF BLOOD
• 1. SECRETORY (HORMONES AND ENZYMES)
• 2. EXCRETORY (WASTE PRODUCTS EG. NON-
PROTEIN NITROGENS/NPN’S)
• 3. NUTRITIVE (VITAMINS AND MINERALS)
• 4. RESPIRATORY (HEMOGLOBIN, MYOGLOBIN)
• 5. IMMUNOLOGIC (ANTIBODIES, WBC’s)

HEMATOLOGY
(INTRODUCTION)
• ANTICOAGULANTS (ADDITIVES WHICH PREVENTS BLOOD
FROM CLOTTING)
• ANTICOAGULANT COLOR OF ACTION
• RUBBER
• STOPPER
• HEPARIN GREEN ACTS ON
• (BLOOD GASES) ANTITHROMBIN-III,
• THROMBOPLASTIN
• CITRATE LIGHT REACTS WITH Ca++
• (COAGULATION BLUE
• STUDIES)
• ETHELENEDIAMINE- LAVENDER CHELATES/SEQUES-
• TETRAACETIC ACID/EDTA TERS CALCIUM
• (COMPLET BLOOD
• COUNT)

HEMATOLOGY
(INTRODUCTION)
• ANTICOAGULANTS (ADDITIVES WHICH PREVENTS BLOOD
FROM CLOTTING)
• ANTICOAGULANT COLOR OF ACTION
• RUBBER
• STOPPER
• OXALATE BLACK REACTS WITH Ca++
• (ERYTHROCYTE
• SEDIMENTATION RATE
• FLUORIDE GRAY REACTS WITH Ca++
• (GLUCOSE)

HEMATOLOGY
(INTRODUCTION)
• OTHER COLORS
• ORANGE/GRAY/YELLOW = THROMBIN =
BLOOD CHEMISTRY
• TAN = HEPARIN/EDTA = LEAD ANALYSIS
• ROYAL BLUE = HEPARIN/EDTA = TOXICOLOGY,
TRACE METALS, NUTRITIONAL ANALYSIS

HEMATOLOGY (INTRODUCTION)
• METHODS OF BLOOD COLLECTION/PHLEBOTOMY
1. VENIPUNCTURE (VENOUS BLOOD: DARK RED)
2.ARTERIAL PUNCTURE (ARTERIAL BLOOD: BRIGHT RED)
3. CAPILLARY/ARTERIOLAR PUNCTURE
(CAPILLARY/ARTERIOLAR/PERIPHERAL BLOOD: BRIGHT
RED)
PALMAR SURFACES OF THE MIDDLE OR RING FINGER
PLANTAR SURFACES OF THE HEEL OR BIG TOE (NEWBORN)
FREE MARGIN OF THE EARLOBE

• TYPES OF BLOOD SPECIMENS
• 55% FLUID, 45% BLOOD CELLS
• 1. WHOLE BLOOD:
• HEMATOLOGY: RBC, WBC, PLATELET COUNTS, Hemoglobin
(Hb), HEMATOCRIT (Hct)
• BLOOD GLUCOSE (GLUCOMETER),
• BLOOD GASES: O2, CO2 BLOOD PH

2. PLASMA: (LIQUID PORTION OF UNCLOTTED BLOOD
WITH FIBRINOGEN)
• CLEAR, LIGHT YELLOW TO SLIGHTLY HAZY
• SELECTED BLOOD CHEMISTRY
– FLUORIDE (GLUCOSE)
– HEPARIN (COAGULATION STUDIES)

HEMATOLOGY (INTRODUCTION
3. SERUM: (LIQUID PORTION OF CLOTTED BLOOD WITHOUT
FIBRINOGEN)
ROUTINE AND SPECIAL BLOOD CHEMISTRY
( HORMONES, DRUGS, ANTIGENS, ANTIBODIES)
FASTING (8-10 HOURS) = CLEAR, PALE YELLOW
NON-FASTING: TURBID OR MILKY (CHYLOMICRONS)
ICTERIC: DARK YELLOW (HIGH BILIRUBIN)

HEMATOLOGY
(INTRODUCTION)
• PROBLEM SITES IN PHLEBOTOMY
– 1. BURNS, SCARS AND TATOOS (IMPAIRED
CIRCULATION)

HEMATOLOGY
(INTRODUCTION)
PROBLEM SITES IN PHLEBOTOMY
– 2. DAMAGED VEINS: SCLEROSED (HARDENED),
THROMBOSED (CLOTTED)= OCCLUDED AND LACK
RESILIENCY

HEMATOLOGY
(INTRODUCTION)
• PROBLEM SITES IN PHLEBOTOMY
• 3. EDEMATOUS AND CYANOTIC AREAS (FALSE
DECREASED CELL COUNTS)

HEMATOLOGY
(INTRODUCTION)
– PROBLEM SITES
– 4. MASTECTOMY (STOPPAGE OF LYMPH FLOW:
SWELLING)

• PROBLEM SITES
• 5. HEMATOMA (SWELLING OR MASS OF
BLOOD IN SKIN DUE TO WRONG PUNCTURE)
– FRAGILE VEINS
– LARGE NEEDLE
– HITTING THROUGH AND THROUGH
– PARTIAL INSERTION OF THE NEEDLE
– EXCESSIVE BLIND PROBING
– REMOVING NEEDLE WITH TOURNIQUET
– INSUFFICIENT PRESSURE AFTER COLLECTION

• HEMOCYTOMETRY (COUNTING OF BLOOD CELLS)
• 1. AUTOMATED (MACHINES)
• EG. COULTER COUNTER (COULTER
ELECTRONICS)
• CELL DYNE 3000 (ABBOTT DIAGNOSTICS)
• COBAS HELIOS (ROCHE)
• E-5000, K-1000, NE-8000,SE-9000 (SYSMEX)
• COMPLETE BLOOD COUNT/CBC INCLUDES: RBC, WBC,
• PLATELETS, DIFFERENTIAL COUNT, HEMOGLOBIN,
HEMATOCRIT, RED BLOOD CELL INDICES
• 2. MANUAL (MICROSCOPE, CENTRIFUGE,
• SPECTROPHOTOMETER)
• COMPLETE BLOOD COUNT INCLUDES: WBC,
• DIFFERENTIAL COUNT, HEMOGLOBIN, HEMATOCRIT

• METHODS AND PRINCIPLES OF AUTOMATED CELL COUNTERS
• 1. ELECTRICAL IMPEDANCE
• CELLS ELECTRODE
• (RBC’s) APERTURE
• (WBC’s) APERTURE
• (PLATELETS) APERTURE
• VOLTAGE PULSE
•
• COUNT
• X-AXIS = PULSE
• VOLUME DISTRIBUTION
• Y-AXIS = CELL SIZE (HISTOGRAM)

• 2. FLOW CYTOMETRY/LIGHT SCATTERING
• HYDRODYNAMIC FOCUSING
• LASER RBC’S FORWARD
• WBC’s ANGLE SCATTER (0)
• PLATELETS
• COUNT
• PHOTODIODE
• DETECTOR
• COMPUTER
• RIGHT ANGLE
• SCATTER (90)
• PHOTOMULTIPLIER DETECTOR

• 2. FLOW CYTOMETRY/LIGHT SCATTERING:
• FLOW CELL (PASSAGE OF CELLS WHICH IS MADE
UP OF QUARTZ)
• FLUOROCHROMES/FLUORESCENT DYES ARE
USED FOR BETTER DIFFERENTIATION
• TYPES OF LIGHT SCATTERING
• REFLECTION: BENDING OF LIGHT DUE TO
CHANGE OF
SPEED LARGE ANGLES
• DIFRACTION: BENDING AROUND CORNERS
(SMALL
ANGLES)
• REFRACTION: TURNING BACK BY THE
SURFACE

• METHODS AND PRINCIPLES OF AUTOMATED CELL
COUNTERS
• HEMOGLOBIN DETERMINATION: SPECTROPHOTOMETRIC
METHOD USING:
• POTASSIUM CYANATE, POTASSIUM FERRICYATE, SODIUM
BICARBONATE (COULTER, ROCHE, ABBOTT)
• SODIUM LAURYL SULFATE (SYSMEX)
• HEMATOCRIT: DERIVED FROM RBC COUNT
• 1 % Hct = 42, 000 RBC’s

• RBC INDICES: COMPUTED BY:
• MCV (MEAN CORPOSCULAR VOLUME)
• = Hct/RBC IN MILLIONS X 10 (80-100 FEMTOLITERS/FL)
• MCH (MEAN CORPOSCULAR HEMOGLOBIN)
• = Hb/RBC IN MILLIONS X 10 (27-32 PICOGRAMS/ PG OR
MICROMICROGRAMS/UUG)
• MCHC (MEAN CORPOSCULAR HEMOGLOBIN
CONCENTRATION) = Hb/Hct X 100% (33-36%)
• Hb X 3 = Hct AND Hct/3 = Hb (NOT APPLICABLE
• FOR NORMOCYTIC HYPOCHROMIC ANEMIA) RULE OF 3

• METHODS AND PRINCIPLES OF AUTOMATED CELL
COUNTERS
• RBC INDICES: COMPUTED BY:
• RDW (RED CELL DISTRIBUTION WIDTH) = SD/MCV OF
RED CELLS IN A GIVEN POPULATION
• PDW (PLATELET DISTRIBUTION WIDTH) = SD/MCV OF
PLATELETS IN A GIVEN POPULATION

• 3. CENTRIFUGAL ANALYSIS
• DO NOT COUNT CELLS BUT JUST ANALYZE RBC AND BUFFY
COUNT IN % EG. QBC ANALYZER (BECTON DICKINSON)
• ACRIDINE ORANGE DYE IS USED
• LAYERS FORMED AFTER CENTRIFUGATION
• PLASMA
• GRANULOCYTES
• PLATELETS
EXPANDED RBC’S
• NON-GRANULOCYTES
• PACKED RBC’S

• MANUAL HEMOCYTOMETRY (HEMACYTOMETER, THOMA
PIPETS (RBC & WBC), MICROSCOPE, SPECTROPHOTOMETER
(Hb), CENTRIFUGE (Hct)
• TYPES OF HEMOCYTOMETER
• CLOSED: THOMA-ZEISS
• OPEN: SPENCER, BURKER, LEVY, LEVY-HAUSSER
• ADDIS/EXTON (URINALYSIS)
• PETROFF (BACTERIA)
• THOMA
• TUERK
• FUCHS-ROSENTHAL
• BASS-JONES
• NEUBAUER
• IMPROVED NEUBAUER

• MANUAL HEMOCYTOMETRY
• .1 MM THICK
• NUMBER OF SQUARES AREA
• 9 LARGE SQUARES 9 MM2
• 4 CORNER LARGE SQUARES (WBC) 1 MM2 X 4 = 4 MM2
• 16 INTERMEDIATE SQUARES 1/16 MM2 =
• .0625 MM2
• 1 CENTRAL LARGE SQUARES (RBC) 1 MM2
• 25 INTERMEDIATE SQUARES 1/25 = .04 MM2
• 16 SMALL SQUARES 1/16 X 25 =
• .0025 MM2

• NUMBER OF SQUARES AREA
• 1 CENTRAL LARGE SQUARES (RBC) 1 MM2
• 5 INTERMEDIATE SQUARES .04 x 5 = .2 MM2
• 80 SMALL SQUARES (5 X 16) .0025 X 80 =
• (USED IN RBC) .2 MM2
• 4 LARGE SQUARES (NOT USED) 1 MM2 X 4 =
• 4 MM2

• WBC COUNT = CELLS COUNTED X DEPTH CORRECTION
FACTOR X DILUTION FACTOR/ AREA FACTOR
• DEPTH CORRECTION FACTOR = 10
• THICKNESS OF HEMACYTOMETER COUNTING CHAMBER
• = .1 MM3 X 10 = 1 MM2
• DILUTION FACTOR = VOLUME OF BULB (10)/VOLUME OF
BLOOD (.5) = 20
• AREA FACTOR = 4 (INTERMEDIATES SQUARES)
• WBC = CELLS COUNTED X 10 X 20 = 50 (MULTIPLICATION
• 4 FACTOR)
• WBC = CELLS COUNTED X 50 = CELLS/MM3

• RBC COUNT = CELLS COUNTED X DEPTH CORRECTION FACTOR
X DILUTION FACTOR/ AREA FACTOR
• DEPTH CORRECTION FACTOR = 10
• THICKNESS OF HEMACYTOMETER COUNTING CHAMBER
• = .1 MM3 X 10 = 1 MM2
• DILUTION FACTOR = VOLUME OF BULB (100)/VOLUME OF
BLOOD (.5) = 200
• AREA FACTOR = 5/25 (INTERMEDIATES SQUARES)
RBC = CELLS COUNTED X 10 X 200 = 10,000 (MULTIPLICA-
• 1/5 OR .2 FACTOR)
• RBC = CELLS COUNTED X 10,000 = CELLS/MM3

• MANUAL HEMOCYTOMETRY ERRORS
• 1. RANDOM/HUMAN
• 2. STANDARD/SYSTEMATIC (EQUIPMENT)
• 3. INHERENT/FIELD (RANDOM SETTLING OF CELLS:
POISSON’S LAW)
• RBC DILUTING FLUIDS
• 1. HAYEM’S + HgCl
• 2. GOWER’S + GLYCERINE
NaCl, NaSO4, DISTILLED H2O
• 3. TOISSON’S + METHYL VIOLET
• 4. BETHEL’S + SODIUM THIOSULFATE
• 5. FORMOL CITRATE/DACIE’S (BEST)
• 6. NORMAL SALINE SOLUTION

• WBC DILUTING FLUIDS
• 2-3 % HAc/CH3COOH WITH METHYL VIOLET (TO
DIFFERENTIATE FROM RBC DILUTING FLUID)
• 1% HCl WITH METHYL VIOLET (TO DIFFERENTIATE FROM RBC
DILUTING FLUID)

• (>10 NRBC’S/HPF)
• CORRECTED WBC/CWBC
• IF > 10 NUCLEATED RBC’S/NRBC’S (IMMATURE)
100 WBC’s IN THE DIFFERENTIAL COUNT
• LONG METHOD
• CWBC =
• UNCORRECTED COUNT X NO. OF NRBC’S
100 + NO. OF NRBC’S
• SHORT METHOD
• CWBC =
• 100________X UNCORRECTED COUNT
• 100 + NRBC’S

• DIFFERENTIAL COUNT
• COUNTING VARIOUS TYPES OF WBC’S (IN %) IN A BLOOD
SMEAR
• BLOOD SMEAR MUST:
• OCCUPY 1/2 TO 2/3 OF THE SLIDE
• HAVE FEATHERY EDGE
• HAVE NO HOLES
• HAVE NO OVERLAPPING CELLS
• HAVE GRADUAL TRANSITION FROM THICK TO
THIN
• STAINED BY: WRIGHT’S OR GIEMSA (MB, EOSIN)

• MAKING A BLOOD SMEAR FOR DIFFERENTIAL COUNT
• 1. 2-SLIDE METHOD
• 2. SLIDE AND COVERSLIP METHOD
• 3. 2-COVERSLIP METHOD
• FACTORS AFFECTING THE QUALITY OF THE SMEAR
• 1. SIZE OF THE DROP OF BLOOD
• 2. ANGLE (30 DEGREES)
• 3. SPEED OF SPREADING
• 4. PRESSURE ON THE SLIDE
• 5. SURFACE OF THE SLIDE AND
• SPREADER

• DIFFERENTIAL COUNT
• METHODS OF COUNTING:
• 1. 2 FIELD MEANDER
• 2. 4-FIELD MEANDER
• 3. EXAGERRATED BATTLEMENT
• 4. STRIP DIFFERENTIAL/CRENELATION
•

• REFERENCE VALUES FOR COMPLETE BLOOD COUNT
• RBC MALE = 4.5-5.9 X 1012 CELLS/LITER OR
• 4,500,000 - 5,900, 000 CELLS/ MM3
• FEMALE = 4.0-5.2 X 1012 CELLS/LITER OR
• 4,000,000 - 5,200, 000 CELLS/ MM3
• Hb MALE = 13.5-17.5 GRAMS/DECILITER (135-175
GRAMS/LITER)
• FEMALE = 12-16 GRAMS/DECILITER (120-160
GRAMS/LITER)
• Hct MALE = .41-.53 LITER/LITER OR 41-53 %
• FEMALE = .36-.46 LITER/LITER OR 36-46 %

• REFERENCE VALUES FOR COMPLETE BLOOD COUNT
• RBC INDICES
• MCH = 26-34 PICOGRAMS(PG) OR
• MICROMICROGRAMS(UUG)
• MCV = 80-100 FEMTOLITERS/FL
• MCHC = 31-37 %
• PLATELET COUNT = 1 X 109 TO 3 X 109 CELLS/LITER OR
100,000-300,000 CELLS /MM3
• RETICULOCYTE/RETICS COUNT = ADULTS = .5-1.5 %
• INFANTS = 2-6 %

• REFERENCE VALUES FOR COMPLETE BLOOD
• WBC MALE AND FEMALE
• 4.5-11 X 10X9 OR 4,500 -11,000
CELLS/MM3
• DIFFERENTIAL COUNT (PRE 200 OR 100 CELLS)
• NEUTROPHILS OR 47-79.5 %
• SEGMENTERS
• LYMPHOCYTES 12.5-40 %
• MONOCYTES 2-11 %
• EOSINOPHILS 0-7.5 %
• BASOPHILS 0-2 %
• BAND/STAB/STAFF 0-5 %
• PLATELETS MUST BE ALSO ESTIMATED (8-10/OIF)

• ESTIMATED WBC AND DIFFERENTIAL COUNT
• WBC/HPF WBC/LITER
• 2-3 4 X 103 TO 7 X 103
• 4-6 7 X 103 TO 10 X 103
• 7-10 10 X 103 TO 13 X 103
• 11-20 13 X 103 TO 18 X 103

• COMMON GREEK AND LATIN PREFIXES USED
• A/AN = LACK, WITHOUT, ABSENT, DECREASED
• ANISO = UNEQUAL. DISSIMILAR
• CYT = CELL
• DYS = ABNORMAL, DIFFICULT, BAD
• ERYTHO= RED
• FERR = IRON
• HEMO/HEMATO = PERTAINING TO BLOOD
• HYPO = BENEATH, UNDER, DEFICIENT
• HYPER = ABOVE, BEYOND, EXTREME
• ISO = EQUAL, ALIKE, SAME
• LEUKO = WHITE

• COMMON GREEK AND LATIN PREFIXES USED
• MACRO = LARGE, LONG
• MEGA = LARGE, GIANT
• META = AFTER, NEXT, CHANGE
• MICRO = SMALL
• MYELO = FROM BONE MARROW, SPINAL CORD
• PAN = ALL, OVERALL, ALL-INCLUSIVE
• PHLEB = VEIN
• PHAGO = EAT, INGEST
• POIKILO = VARIED, IRREGULAR
• POLY = MANY
• SCHIS = SPLIT
• SCLER = HARD
• SPLEN = SPLEEN
• THROMB = CLOT
• XANTH= YELLOW

• COMMON GREEK AND LATIN SUFFIXES USED
• CYTO = CELL
• EMIA = BLOOD
• ITIS = INFLAMMATION
• LYSIS = DESTRUCTION/DISSOLVING
• OMA = SWELLINNG/TUMOR
• OPATHY = DISEASE
• OSIS = ABNORMAL INCREASE
• PENIA = DEFICIENCY, DECREASE
• PHIL = ATTRACTED TO, AFFINITY FOR
• PLASIA = CELL PRODUCTION OR REPAIR
• POIESIS = CELL PRODUCTION, FORMATION AND
DEVELOPMENT

• COMMON GREEK AND LATIN PREFIXES AND
SUFFIXES COMBINATIONS USED
• ANISOCYTOSIS = VARIATION IN CELLS SIZE
• POIKILOCYTOSIS = VARIATION IN CELL SHAPE
• APLASIA = ABSENCE OF CELLULAR PRODUCTION
• DYSMYELOPOIESIS = ABNORMAL DEVELOPMENT OF
MARROW CELLS
• PANMYELOSIS = AN ABNORMAL INCREASE IN ALL
MARROW CELLS

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