Heme Lab 2 Test Review

Qualitative methods of hemoglobin assessment.

Screen:

• Used first for most common abnormal hemoglobin

Sickle Solubility test to detect ONLY Hgb S

• Negative sickle solubility test only rules out the presence of HgbS; other abnormal Hgb, may be present and may be detected and confirmed by Hgb electrophoresis

Describe the principle of the sickle solubility test.

• Detects both homozygous (S/S, “disease“) and heterozygous (S/A, “trait or S/C)

• Saponin lyses RBC; Hgb released

• Sodium hydrosulfite reduces releases Hgb

How to interpret Sickle cell solubility test?

• If Hgb S is present it is insoluable in reduced state; forms cloudy, suspension in tube

• A reader card is placed behind the tube

  • If the lines cannot be seen the test is positive

  • If the lines can be seen the test is negative

Explain the limitations of a sickle solubility test.

False negatives:

• Marked anemia: insufficient quantities of Hgb S

• Infants: still have elevated Hgb F (Not Hgb S) production

• Post-transfusion: Hgb S may be diluted

False positives:

• Polycythemia: too much Hgb, not enough reagent

• Dysglobulinemias: abnormal proteins can cause turbidity

Note: Hgb C harlem and Hgb C georgetown also give positive reactions

Discuss the principle of hemoglobin electrophoresis.

• EDTA- anticoagulated blood centrifuged

• RBCs are washed and lysed

• Hemolysate and controls applied to agarose gel

• Placed in electrophoresis chamber for ~25 minutes

• Gel stained and examined

• Can quantify Hgb present using densitometry

Compare and Contrast hemoglobin electrophoresis on cellulose acetate and citrate agar gel.

Alkaline (Cellulose Acetate) pH 8.6:

• Hgb molecules have a negative charge; migrate towards the anode

• Amino acids substitutions in hemoglobin variants alter net charge and mobility

Acid (Citrate agar) pH 6.2:

• Hgb molecules separate into bands that behave differently at an acid pH

• Used to differentiate Hgb variants that migrate together on cellulose acetae (i.e. HgbS from HgbD and HgbG, HgbC from HgbE)

Define hematocrit

Volume of packed RBCs in a given volume of blood

AKA packed cell volume

Reference range: 36-52%

Define hemoglobin

Red pigmented, iron- and oxygen-containing protein created and housed in RBCs

What is the absorbance value of cyanmethemoglobin?

540 nm

What is the cyanmethemoglobin method?

• Modern analyzers determine Hgb concentration using spectrophotometry to measure the amount of light that a sample absorbs at a certain wavelength

• Performed in WBC chamber of the hematology analyzer

• Whole blood is diluted in a fluid that:

  • Contains both potassium ferricyanide and potassium cyanide

  • Lyses the RBCs, releasing Hgb into the solution

• Fe 2+ of the Hgb molecule is oxidized by potassium ferricyanide to Fe 3+, forming methemoglobin

• Potassium cyanide then form cyanmethemoglobin, causing a red color change

State possible sources of error in measuring hemoglobin concentration, including potential impact on results.

• Several physiologic conditions may interfere:

  • Extremely high WBC count

  • Lipemia

  • Target cells and cells containing Hgb S and/or C

  • Heavy smokers — carboxyHgb

Compare automated and manual methods used for hematocrit determinations.

Manual method:

• Capillary tube is filled with anticoagulated whole blood

• Centrifuged in a microhematocrit centrifuge at high speed for a short time

• Measured directly

• Value slightly higher than automated

Automated method:

• Measured indirectly by calculation based on RBC indices

• HCT = (RBC (millions/uL) x MCV (fL)) / 10

List the steps in the ESR procedure.

• The test is performed at a room temperature of 18-25 C within 4 hours of collection

• Each ESR test is set up separately so that the result can be read in one hour

• Prior to setting up, thoroughly check specimen with applicator sticks to ensure that no clot is present. Write your name on the clear ESR vial, being careful not to obscure the fill line

• Remove the stopper (pink cap) of the prefilled vial (0.2 mL of 3.8% Na Citrate is used as diluent).

• Gently invert purple top a minimum of ten times to thoroughly mix. Immediately proceed to next step