HEMA (CEU LAB) Flashcards by LEDESMA, Anne Gelika

science of blood, its nature, functions and diseases

Hematology

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gives information of the anatomy and physiology of blood and laboratory methods by which this information is commonly obtained

Hematology

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suspension of RBC, WBC, Platelets, and blood dust or hemokoniae in a fluid of variable composition, the plasma

Blood

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fluid of variable composition where RBC, WBC, platelets, and blood dusts are suspended

plasma/serum

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blood dust

hemokoniae

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blood SG

1.052 - 1.061

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blood pH

slightly alk (7.3 - 7.5)

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total blood volume in an ADULT (in L and %)

5-6 L
7-8% of total BW

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% of formed elements in the blood

45%

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% of fluid portion of the blood

55%

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Formed elements of the blood

RBC
WBC
PLT

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Composition of the fluid portion of the blood

90% water
10% protein, carbo, vitamins, hormones, enzymes, lipids, salts

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major proteins in the blood

albumin
globin
fibrinogen

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2 types of blood collection

skin puncture
venipuncture

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sites for skin puncture

finger
earlobe
plantar surface of the great toe or heel

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enumerate process of skin puncture

rub the part well with 70% alc
allow to dry
make a quick and good stab
use the NEXT DROP of blood for examination

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enumerate venipuncture process

preparation of materials
tourniquet application
vein selection
sterilization
needle insertion
withdrawal of blood
tourniquet release
withdrawal of needle
prevent further bleeding using dry cotton

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2 types of blood liquid portion

serum
plasma

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liquid portion without fibrinogen

serum
(consumed during clotting)

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aka Heller and Paul’s or Wintrobe mixture

Ammonium and Potassium oxalates; 3:2 (double oxalates)

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Heller and Paul’s or Wintrobe mixture composition that cause SWELLING

ammonium salts

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Heller and Paul’s or Wintrobe mixture composition that cause SHRINKAGE

potassium salts

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Composition of Heller and Paul’s or Wintrobe mixture with corresponding measurement

Ammonium oxalate - 1.2 g
Potassium oxalate - 0.8 g
Distilled water - 100.0 mL

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EDTA means

Dipotassium or Disodium salt of Ethylenediaminetetraacetic acid

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prevents calcium from ionizing by chelation

Dipotassium or Disodium salt of Ethylenediaminetetraacetic acid (EDTA)

Needed EDTA per 1 ml of blood

1-2 mg

Importance of CBC

Indicator of body's ability to fight diseases Monitor drug effects and radiation therapy Indicator of progress in diseases (infection, anemia)

Included in CBC

Hemoglobin Concentration (Hemoglobin) Erythrocyte Volume Fraction (Hematocrit) Erythrocyte Number Conc. (RBC count) Leukocyte Number Conc. (WBC count) Leukocyte Type Number Fraction (Diff. Count) Thrombocyte Number Conc. (Platelet Count) Red Blood Cell Indices

iron-bearing protein contained within the erythrocytes in normal blood

Hemoglobin

represents the colored components in hemoglobin

heme

complex protein in hgb

globin

Carrier of oxygen from the lungs to the tissues where it readily releases this oxygen to the tissue and then returns to the lungs to combine with more oxygen. This respiratory function of hemoglobin is vital to the life of the cells.

Hemoglobin

Methods of Estimation of Hemoglobin

Copper Sulfate or Specific Gravity Method Gasometric Method (Oxygen Capacity Method) Chemical Method (Iron Content Method) Colorimetric Method

aka Gasometric Method

Oxygen Capacity Method

aka Copper Sulfate method

Specific Gravity method

aka Chemical method for HGB

Iron Content method

2 types of colorimetric method for hgb

Visual comparison Photoelectric

Under visual comparison of HGB colorimetric method

Direct matching Acid Hematin Alkali Hematin

Direct Matching methods for HGB

Tallqvist Dare

Acid hematin methods for HGB

Sahli's Hellige Haden-Hausser Sahli-Adams Haldane Osgood Newcomer

Alkali hematin methods for HGB

Clegg & King

Photoelectric methods for HGB

Oxyhemoglobin method Cyanmethemoglobin

Amount of Drabkin's reagent to blood

Drabkin's = 5 mL Blood = 20 uL

HGB measurement process

Place 5 ml of Drabkin's reagent in a small amber colored vial Suck 20 ul of blood using sahli's pipette. Transfer 20 ul of blood in a small amber colored vial with drabkin's reagent Stand for 10 minutes. Allowing the RBCs to hemolyse. Measure density of the solution at 540 nm, using water as blank Obtain hemoglobin value from a calibration curve prepared with the aid of standard

Wavelength of HGB reading

540 nm

aka Hematocrit

Packed Cell Volume Erythrocyte Volume Fraction

one of the accurate and valuable hematologic investigations

Hematocrit

Methods of Hematocrit Determination

Macrohematocrit by Wintrobe Microhematocrit method

thick flat bottom tube 11.5 cm long, 3 mm internal bore, graduated 0-100 mm. The left hand side graduation (descending) is for ESR and the right side graduation (ascending) is for hematocrit

Wintrobe tube

WINTROBE TUBE: right hand side graduation (ascending) is for ____

HCT

Under Macrohematocrit by Wintrobe

Wintrobe tube Brays Van Allen's Stanford-Magath Haden's modification

graduation of Wintrobe tube

0-100 mm

length of Wintrobe tube

11.5 cm

originally designed for capillary blood collection, using capillary tube, either heparinized or non-heparinized

Adam's

Under Microhematocrit method

Adam's

internal bore of WIntrobe tube

3 mm

WINTROBE TUBE: left hand side graduation (descending) is for ____

ESR

measure of capillary tube left unfilled

10 mm

Centrifugation time and speed for microhematocrit method

5 minutes at high speed

process of enumerating blood cells like leukocytes, erythrocytes and thrombocytes.

Hemocytometry

made up of thick rectangular glass slides

counting chamber

Cells touching half way in and half way out of the ___________ boundary lines of the square are counted.

upper and left

Counting chambers

Improved Neubauer Fuchs-Rosenthal

Cells touching half way in and half way out of the _______ boundary lines of the square are not counted.

lower and right

Double lines (_____ line is the boundary) Triple lines (______line is the boundary)

outer middle

Remedy for irregularly distributed cells in chamber

re-charge

determination of the number of red blood cells present in 1 ul of blood.

Erythrocyte Count

Mark of thoma pipette for RBC count

101

Dilution of thoma pipette for RBC count

1:200

Bead color of thoma pipette for RBC count

Red

Bulb size of thoma pipette for RBC count

Big

Bore size of thoma pipette for RBC count

small

determination of the number of white blood cells present per 1 ul of blood

Leukocyte Count

Diluting fluids for RBC count

Dacies or formol citrate solution Hayem's diluting fluid Gower's solution Toisson's fluid Bethell's fluid NSS or Physiologic Salt Solution 3.8 Sodium Citrate

Mark of thoma pipette for WBC count

Bead color of thoma pipette for WBC count

White

Dilution of thoma pipette for WBC count

1:20

Bulb size of thoma pipette for WBC count

Small

Bore size of thoma pipette for WBC count

Big

Diluting fluid for WBC count

2-3% acetic acid

Computation for RBC ct

Number of RBC counted X 10,000 = RBC/uL of blood

Computation for WBC ct

Number of WBC counted X .05 = WBC X 10^9/L

Automated Method of Blood Smear Preparation

Spun Smear Smear prepared in Miniprep (wedge/push smear)

size of blood drop used for blood smear

2-3 mm diameter

Blood Smear Manual methods

2 glass slide method/wedge/push method Coverglass method = Ehrlich's method Coverglass and slide method = Beacom's method

angle of slide spreader during blood smear

40 degree

Factors that determine thickness or thinness of smear

Size of blood drop Large angle between slide and spreader Speed of spreader Pressure of spreader over slide

Criteria of a Good Smear

Gradual transition from thick to thin smear. Smear must cover 3⁄4 of the slide. Must have a smooth even surface, free from ridges, waves and holes. No overlapping of cells.

PBS staining method

Solution 1 (methanol) — fixative (10 dips) Solution 2 (eosin) — acid stain (10 dips) Solution 3 (methylene blue) — basic stain (10 dips) Solution 4 — buffer (15 dips)

Other staining method

Wright's stain Leishman's stain Jenner-Giemsa stain May-Grunwald-Giemsa stain Giemsa stain

consists of the enumeration of the relative proportions (percentages) of the various types of white blood cells as seen in stained films of peripheral blood

Differential Count

WAYS OF SCANNING SMEARS FOR DIFFERENTIAL COUNT

Strip or Horizontal Method Crenellation Method Exaggerated Battlement Method Two Field Meander Method Four Field Meander Method

all the cells in longitudinal strips from head to end or tails of the smears are counted

Strip or Horizontal Method

cells are counted from the upper part of the smear, the lower part, then sideways, then to the upper part

Crenellation Method

one begins at one edge of the smear and counts all cells advancing inward from 1/3 of the width of the smear, to a line parallel to the edge, out to the edge to along the edge for an equal distance before turning Inward again and again

Exaggerated Battlement Method

smear is divided Into two fields. Similar to exaggerated battlement method. Fifty cells for each of the two fields are counted

Two Field Meander Method

more accurate than two field meander since the four areas where the cells are counted represents the entire smear

Four Field Meander Method

Stop or retard the action of foreign materials

Neutrophil

9-12 um in diameter Pink to rose-violet Normally 2-5 lobes connected with thin filaments

Neutrophil

Release of potent oxidants to destroy helminth parasites

Eosinophil

Anti-inflammatory cell

Eosinophil

Produces heparin & histamine against allergies

Basophil

9-12 um in diameter Reddish-orange granules Two lobes coarse, clumped chromatin pattern

Eosinophil

They exhibit chemotaxis and some phagocytic activity

Basophil

10-16 um in diameter Slightly pink to colorless cytoplasm and dark purple to blue black granules Unsegmented or bilobed, rarely has three or four lobes

Basophil

Defense mechanism Removal of damaged and old cells, plasma protein, & plasma lipids

Monocyte

Provide for the recognition and elimination of foreign pathogens, proteins and altered cells from the body

Lymphocyte

14-20 um in diameter Blue gray outline, maybe irregular because of the presence of pseudopods, azurophilic granules, giving a ground glass appearance Round, kidney shape. No nuclei are visible, chromatin is fine and lacey arranged in skein-like strand

Monocyte

Maintain the body's resistance to these offending agents

Lymphocyte

Pale to moderate blue Round or oval in shape

Lymphocyte (can be small, medium, large)

Platelet count methods

Toncatin Method using the Rees and Ecker diluent Phase Contrast Microscopy using 1% ammonium oxalate diluting fluid

Used for phase contrast microscopy of platelets

1% ammonium oxalate diluting fluid

contains citrate-formaldehyde buffer with brilliant cresyl blue as a platelet stain for light microscopy

Toncatin Method using the Rees and Ecker diluent

average volume of red cells in a given volume of blood

Mean Cell Volume (MCV)

MCV formula

Hct x 10 /RBC/L

SI unit of MCV

femtoliter

average hemoglobin content of the red blood cell

Mean Cell Hemoglobin (MCH)

MCV normal value

80-100 fL

SI unit of MCH

pictogram

MCH formula

Hgb (g/dL) x 10 / RBC

MCH normal value

27-31 pg

average hemoglobin concentration in a given volume of packed erythrocytes

Mean Cell Hemoglobin Concentration (MCHC)

SI unit of MCHC

grams of hemoglobin per liter

MCHC formula

Hgb x 100 / Hct

MCHC normal value

31-36 g/dL

Principle of Hematology Analyzer for RBC, WBC, Platelets

Electrical Impedance/Direct Current Optical Count/Flow Cytometry

measure of the total opposition to current flow in an alternating current circuit made up of two components, ohmic resistance and reactance

Optical Count/Flow Cytometry

Principle of Hematology Analyzer for HGB

Cyanmethemoglobin Modified Methemoglobin Chromogen (cyanide-free)

Hemoglobin measurement is done directly in the WBC cuvette, by spectrophotometry. The light source is an L.E.D. (Light Emitting Diode) at 555 nm. Hemoglobin is detected by formation of Cyanmethemoglobin chromogen when using a lyse solution with cyanide.

Modified Methemoglobin Chromogen (cyanide-free)

Principle of Hematology Analyzer for HCT

Measurement by Summation Automatically derived by the automated analyzer from the RBC count and MCV value.

DIFF COUNT PRINCIPLE size or volume of the particle

Aperture-Impedance/Direct Current

DIFF COUNT PRINCIPLE nuclear size and density, cytoplasmic granularity

Radio Frequency Conductivity

DIFF COUNT PRINCIPLE size and characteristics, like nuclear shape and cytoplasmic granules of a cell

Optical Light Scatter Flow Cytometry

DIFF COUNT PRINCIPLE presence of specific enzyme or protein- dye precipitate

Cytochemistry

DIFF COUNT PRINCIPLE antibody specific for a cell protein

Fluorescence/Immunological Flow Cytometry

Principle of Hematology Analyzer for PLT CT

Impedence / Direct Current Optical Count / Flow Cytometry

Principle of Hematology Analyzer for MCV

DIRECTLY MEASURED

Principle of Hematology Analyzer for MCH

Hgb / RBC × 10

Principle of Hematology Analyzer for MCHC

Hgb / Hct ×100

Principle of Hematology Analyzer for RDW

Histogram Derived

composition of buffy coat

WBC Platelets

site of skin puncture for adults

all fingers

site of skin puncture for babies

plantar surface of heel or great toe

caused by air sucked by syringe --> veins

embolism

not preferred anticoag due to crystallization

double oxalates

used for plasma hgb

oxyhemoglobin

hgb subjected to spectro at 540 nm

cyanmethemoglobin

used capillary tube if hematocrit is to be carried out using anticoagulated tube

nonheparinized blue

used capillary tube for direct prick

heparinized red

allowable difference in 4 quadrants of counting chamber

WBCs are read using this field

low power dark field

effect on cell count of OVERFILLED chamber

decreased count

effect on cell count of UNDERFILLED chamber

increased count

Best RBC diluting fluid

Dacies or formol citrate solution

RBC diluting fluid preventing rouleaux formation

Gower's solution

RBC diluting fluid causing rouleaux formation

NSS

Smear technique for BM aspirate

Coverglass method (Ehrlich's)

Smear technique employed by machines

Coverglass and Slide method (Beacom's)

PBS are read using: a. Bright field b. Dark field

a. Bright field