TRANSCRIBED GEN CAMATO HEMATOLOGY LABORATORY

HEMATOLOGY 1 | LABORATORY

1

X 100

9 Very useful to establish anemia; & other disorder in blood 9 MCH & MCV Hemoglobin, Hematocrit & RBC

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NON-PATHOLOGICAL

PATHOLOGICAL

PATHOLOGICAL

HEMATOCRIT Parallel to hemoglobin Volume occupy by erythrocytes in the given volume of blood Useful in determining erythrocyte indices and for calculation of a

blood volume Useful for total erythrocyte mass determination Useful in establishing anemia

METHODS FOR HEMATOCRIT

1) Macro method Use large volume of blood a) Winthrobe method

Uses Winthrobe tube Flat bottom tube graduated at both side Fill the tube with blood sample using capillary pipet Uses oxalated blood sample Alternative anticoagulant: EDTA Centrifuge the tube with the speed of 3,500rpm for

30minutes Calculation for % Hematocrit

Volume % of Hematocrit = Hematocrit of Packed RBC Hematocrit of White Blood

Reference range: Conventional unit Male: 47 vol.% Female: 43 vol.%

b) Haydens modification Anticoagulant is 1.1% of NaC2O4 (Na Oxalate)

of 3.8% Na Citrate c) Van Allens modification method

1.6% or 1.3% NaC2O4 or 3.8% Na Citrate d) Sanford Magath

1.1% of 1.3% Na oxalate or 3.8% Na citrate e) Brays Method

Heparin is the anticoagulant 2) Micro method

a) Micro Adams method Most popular and simple Uses heparinize capillary tube Mix to avoid coagulation Seal with clay sealer then wax Spin: 10,00012,000 rounds per minute (RPM) for

5minutes Read with micro hematocrit capillary reader

Note: The first drop of blood is usually discarded because it is contaminated with dead epidermal cells and tissue juices.

ESR Erythrocyte Sedimentation Rate Rate of setting of RBC from the plasma after an addition of

anticoagulant Importance to measure rate of fall Associated with the net-surface charge of RBC with normal

surface of charge negative IMPORTANCE OF ESR Use as a good index for determination of a hidden but active

disease such as tuberculosis and carcinoma Measures the suspension stability of the RBC Measures the abnormal concentration of fibrinogen and protein

globulin ESR METHODS

1. Winthrobe Method Uses double oxalate; alternative: EDTA Fill in the capillary pipet, stand for an hour

2. Westergren Method * For RESEARCH purpose

Most sensitive method of ESR determination use for serial studies of chronic diseases such as carcinoma and tuberculosis

Done in 2 readings after an hour and after 2 hours Anticoagulant: 3.8% Na Citrate

3. Brays Method

Anticoagulant: 1.3% Na Oxalate

4. Cutler Method Anticoagulant: 3.8% Na Citrate

5. Micro Method

Especially for neonates a) Landau Smith Method b) Smith Method

CONDITIONS OF ESR

r FASTER RATE ESR Pregnant women Menstruating women Suffering with:

Tuberculosis Cancer Rheumatic fever Malignant lymphoma

r SLOWER RATE ESR Spherocytosis Poikilocytosis Sickle cell anemia Severe Iron Deficiency Anemia (IDA) Thalassemia Jaundice

TRANSCRIBED GEN CAMATO HEMATOLOGY LABORATORY

HEMATOLOGY 1 | LABORATORY

2

ZETA SEDIMENTATION RATIO Determines the suspension stability of RBC that is not

affected by anemia Measures the degree of packing of erythrocyte that occurs

during a 45 degrees cycle of dispension and compaction of RBC in a special capillary tube and special centrifuge: zetafuge

Zetafuge four spin cycle of 400 RPM/45 sec. For 4 mins. Reference Value: 4051%

HEMOCYTOMETRY

Numerical evaluation of the formed elements of blood Estimation of the number of blood cells in a given

volume of blood METHODS FOR HEMOCYTOMETRY * OBSOLETE METHODS; NO LONGER USED

1) Turbidimetry Base on the assumption that the more turbid the solution,

the more cells are present.

2) Microscopic Method * MANUAL METHOD Using pipet method/techniques or test tube dilution

method Principle: blood cells are counted under the microscope

with the use of counting chamber, pipets, and diluting fluid TYPES OF COUNTING CHAMBER

1. Spencer New Improved Neubauer Counting Chamber Open type It has 2 center flat form 1 ruled are divided in 9 primary square (1 sq.mm) 4 large square use for count Central primary square count Depth: 0.1 mm

2. Fuchs Rosenthal Design for CSF analysis Depth: 0.2 mm

3. Speirs Levy

Has 4 center flatforms PIPETS (Automatic and Non-Automatic)

1) Unopette * COMMONLY USED; PREFERRED FOR PLATELET COUNTING [ Microblast capillary pipet that automatically suck just

the right amount of a sample, connected into a plastic container, containing just the right amount of diluting fluid

2) Thoma pipet *MANUAL

CLEANING OF THE PIPETS

1. Wash with water, alcohol and then ether. Air dry 2. Wash with water, acetone. Air dry

RBC Pipette WBC Pipette

Color of Bead Size of Bulb Larger Smaller

Calibration Mark 101 11

Size of Lumen Smaller Larger

TRANSCRIBED GEN CAMATO HEMATOLOGY LABORATORY

HEMATOLOGY 1 | LABORATORY

3

= 20 L

STEPS IN HEMOCYTOMETER A. Pipet Method

1. Suck blood up to 0.5 mark of the pipet 2. Suck diluting fluid up to 101 (for RBC) and 11 (for WBC) 3. Shake pipet to mix 4. Discard first few drops 5. Charge counting chamber 6. Count RBC in 5 intermediate square and WBC in 4 corner

larger square

B. Test tube dilution Method 1. Add 4mL of dilution fluid 2. Shake tube 3. Obtain sample by a capillary tube 4. Charger counting chamber 5. Count in counting chamber

Note: to convert mL to L ex. 0.02 mL x 1000 L 1mL COMPUTATION FOR RBC in Millions/mm3 = RBC count x 10 x 200 x 5 I Wherein: 10 depth correction factor (constant) 200 dilution factor (variable) 5 area correction factor (constant) COMPUTATION FOR WBC in Thousand/mm3 = WBC counted x 10 x 20 4 I Wherein: 10 depth correction factor (constant) 20 dilution factor (variable) 4 area correction factor (constant) CHARACTERISTIC OF IDEAL DILUTING FLUID Isotonic (RBC) Hypotonic (WBC)

9 0.85 -0.9% NaCl 9 to Lyse RBC * To prevent RBC from Shrinking & Swelling

Chead and economical Easy to secure and prepare With preservative action With high specific gravity Stable With buffer action Non-allergenic Non-corrosive

RBC DILUTING FLUID ! Hayems

Initiates mold and rouleaux formations Can stand for a long period of time and no corrosive

effect ! Gowers

Prevents rouleaux formation and precipitation of protein ! Toissons

Initiates mold formation so it should be filtered With high specific gravity With stain so used by beginners since RBC are easily

identified form WBCs whose nuclei stain are blue.

(Continuation RBC dilution) ! Formol citrate (Dacies fluid)

Best RBC diluting fluid With preservative action so NO MOLD formation Cell morphology not altered

! NSS

Used in cases of emergency Ideal to use in case of excessive rouleaux formation or

agglutination. WBC DILUTING FLUID * Function: to LYSE RBC ! 1-3% Acetic acid with Gentian violet ! 1% HCl ! Tuerks

Acetic acid Methyl violet Distilled water

2 TYPES OF AUTOMATED HEMOCYTOMETRY

1) OPTICAL AUTOMATED Principle: blood cells are counted as deflections of light

beams passes to the dark field area of the machine. Example: Fisher autocytometer

2) ELECTRICAL AUTOCYTOMETER Principle: blood cells are counted as changes in voltage

pulse Example: Coulter counter (diluting fluid: Isoton)

Inclusion Composed of Stain Indications

Howell-jolly DNA Wright

Disturbed erythropoietin

Hemolytic Anemia

Megaloblastic anemia

Post-splenectomy

Basophilic stippling RNA

Wright New Methylene

Blue

Thalassemia Lead poisoning

Pappenherimer bodies

Siderotic granules

Denatured precipitated hemoglobin

Supravital Stain

G6PD deficiency Thalassemia Unstable

hemoglobins

Cabots ring Remnants of mitotic spindle Wright Megaloblastic

anemia

Parasites Malaria Babesia Trypanosomes

Wright Parasitic infection

A wise man will hear, and will increase learning; and a man of understanding shall attain unto wise counsels:

- Proverbs 1:5