HEMA_Midterms_lec: Laboratory Evaluation Part 1 (in progress)

COPPER SULFATE / SPECIFIC GRAVITY METHOD

- uses Copper sufate to assess the S.G. of blood

lower

If the blood floats, it has a (lower/higger) specific gravity than copper sulfate

higher

If the blood sinks, it has a (lower/higher) specific gravity than copper sulfate

GASOMETRIC / OXYGEN CAPACITY METHOD

Principle: Hb will combine and liberate a fixed quantity of O₂. The blood is hemolyzed with saponin and the gas is collected and measured in a Van Slyke apparatus

GASOMETRIC / OXYGEN CAPACITY METHOD

- not used for the clinical set up, but only for conducting researches about hemoglobin

- takes a long time

ACID HEMATIN METHOD

Principle: The yellowish-brown end-product will be compared to the color on the comparator block

Oxyhemoglobin Method

- measures plasma hemoglobin

- used in arterial blood gas analysis

- to monitor oxygen levels in patients

Free Hemoglobin

Plasma hemoglobin refers to

Cyanmethemoglobin Method

gold standard and reference method used to measure hemoglobin because it can measure all types of hemoglobin except sulfhemoglobin

Cyanmethemoglobin method

Principle: Blood is diluted in a solution of potassium ferricyanide and potassium cyanide.

<> Cyanmethemoglobin method principle

In cyanmethemoglobin method

Hb -> Methemoglobin -> Cyanmethemoglobin

Potassium ferricyanide

converts Hb to MethHb

Potassium cyanide

converts MethHb to CyanMethHb

Procedure for CyanMethHb Method

Collect Whole Blood: EDTA

1. Aspirate 5mL of Drabkin's rgt into a tube

2. Add 20 uL of Blood

3. Mix and Stand for 5 mins/

4. Transfer to a cuvette and read at 540 nm using a spectrophotometer

- 1 g sodium bicarbonate

- 52 mg potassium cyanide

- 198 mg potassium ferricyanide

- 1,000 ml distilled water

MODIFIED DRABKIN'S REAGENT composition

Male: 13.5-18 g/dL

Female: 12-15 g/dL

Hb Reference range

Male:

Female:

- Age

- Sex

- Altitude of Locality

Factors affecting Hb results

Hyperchromia

increased HB level

- Polycythemia

- Dehydration

- Changing from higher to low altitudes

Hyperchromia can be found in what conditions:

Oligochromia

Decreased hemoglobin level found in anemias

Correction: Store in a brown bottle or in a dark place

> Cover the test tube containing the Drabkin's reagent with carbon paper or foil

Correction:

Sources of Error: Drabkin's reagent is sensitive to light

Correction: Centrifuge the solution and measure the supernatant

> Supernatant - liquid on top after centrifugation

SOE: Increased WBC Count (>20 x 109 /L) and increased Platelet Count (>700 x109 /L) can cause turbidity and a falsely high result

Correction: Add 0.01 mL of the patient's plasma to 5 mL of Drabkin's reagent and used the solution as reagent blank

SOE: Lipemic sample can interfere and can give a false result

Correction: Dilution with distilled water (1:2)

SOE: Hb S and Hb C may be resistant to hemolysis causing turbidity

Correction: Add 0.1 g of potassium carbonate to Drabkin's reagent

SOE: Abnormal globulins found in multiple myeloma (high protein) or Waldenstrom macroglobulinemia may precipitate

- Anemia

- Iron deficiency

- Thalassemia

- Pernicious anemia

- Liver disease

- Hypothyroidism

- Hemorrhage

- Hemolytic anemia

- Systemic diseases

Conditions with Decreased Hb levels

- Polycythemia vera

- Congestive HEart failure

- Chronic Obstructive Pulmonary Disease (COPD)

Conditions with increased Hb levels

Hematocrit/Packed Cell volume

Principle: Anticoagulated whole blood is centrifuged, and the total volume of the red cell mass is expressed as a percentage or a decimal fraction

1. Lipids

2. Plasma

3. Buffy coat

4. Packed RBCs

5. Clay

6. Wax

6 layer of Hematocrit (Top to bottom)

Red Band (heparinized) - used for blood collection during skin puncture

Blue band (not heparinized) - used for collecting blood from EDTA/heparin tubes

2 types of capillary tubes

o Anemia

o Leukemia

o Lymphomas

o Adrenal insufficiency

o Chronic disease

o Acute and chronic blood loss

Decreased values fo Hct may indicate:

- erythrocytosis

- Polycythemia vera

- Shock

Increased Hct values occur in

1. Should be Hypotonic

2. Stains the nucleus of white blood cells

Criteria of Good WBC diluting Fluid

- 2-3% glacial acetic acid

- 1% HCl added with 2 drop of methyl violet or crystal violet

WBC Diluting Fluid composition

True

T/F

Corrected WBC Count: must be corrected if 5 or more nucleated RBCs (NRBCs) are counted on differential count since NRBCs (immature RBCs) present in the sample are not lysed by the diluting fluid and counted as WBC

Hayem's Fluid

- used in the RBC counting experiment in lab

Gower's Solution

prevents rouleaux formation

Toisson's Fluid

- high specific gravity

- stains the WBC

Dacie's Fluid / Formol Citrate Solution

- best diluent

- keeps for a long time and does not alter the shape of the cells

Normal Saline Solution

- used in emergency cases

- used in the presence of rouleaux formation and autoagglutination of cells

- 10 mL 40% formaldehyde

- 990 mL 3% w/v disodium citrate

Dacie's Fluid / Formol Citrate Composition:

- 1 g mercuric chloride

- 4.4 g sodium sulfate anhydrous

Hayem's Diluting Fluid Composition:

- 12.5 g sodium sulfate anhydrous

- 33.3 mL glacial acetic acid

- 200 mL distilled water

Gower's solution

- 1 g sodium Chloride

- 8 g Sodium Sulfate

- 30 glycerin

- 0.25 g methyl violet

- 180 mL distilled water

Toisson's Fluid Composition

- 0.85 g sodium chloride

- 100 mL distilled water

Normal Saline Solution Composition

- 3.8 g sodium citrate

- 100 mL distilled water

3.8% Sodium citrate

1. must be an isotonic solution

2. has a good preservative

3. Does not initiate the growth of molds and yeast

4. High S.G

5. With buffer action

6. Cheap and easy to prepare

Criteria of Good RBC diluting Fluid

normocytic normochromic erythrocytes

Rule of Three is applied to what type of erythrocytes

RBC x 3 = Hb

Hb x 3 = Hct -/+ 3

Note: only applied if Hb and RBC is normal value

Rule of 3

Normal value

Male: 42-54%

Female: 35-49%

Normal value of Hb level

Male:

Female:

4.5-11.5x10^9 /L

Normal value of WBC Count

Male: 4.6-6.0x10^12 /L

Female: 4.0-5.4x10^12 /L

Normal value of RBC Count