Measuring Hemoglobin and Hematocrit

hematocrit

percentage of RBC in blood sample

usually expressed as a percentage of the volume of the whole blood

diagnosing anemia

dehydration

polycythemia

clinical importance of hematocrit

principle of hematocrit

centrifugation separates blood components into plasma, buffy coat, and packed RBCs

RBC layer is measured as a percentage of total blood volume

hematocrit procedure

pill capillary tube with anticoagulated blood

seal one end

centrifuge the sample at 10,000-12,000 RPM for 3-5 minutes

use hematocrit reader to determine the percentage of packed RBCs

micro-hematocrit technique

blood can be either from a capillary puncture or an EDTA tube

make sure EDTA specimen is well mixed

holding hematocrit tube horizontally, touch the top of the tube to the blood and allow it to fill by capillary action, no more than ¾ full —> fill 2 tubes

seal one end of capillary tube with Crit-o-seal

centrifuge tubes in a micro-hematocrit centrifuge —> make sure sealed ends of capillary tubes face outward

screw on cover of centrifuge and close the lid —> centrifuge the tubes for 5 minutes

read hematocrit % using a hematocrit reader —> reults must agree within ± 2% (average of 2 is reported)

falsely lowers results due to over packing of RBCs

if capillary tube is centrifuged for too long

falsely elevates results due to under packing the RBCs

if capillary tube is not centrifuged enough

hematocrit

adequate centrifugation is necessary for a correct _______

EDTA

WB

the ratio of _____ to _____ should be adequate

cell shrinkage

too much EDTA in whole blood sample can cause ?

40-52%

normal adult male hematocrit percentages

37-47%

normal adult female hematocrit percentages

35-45%

normal children hematocrit percentages

hemoglobin

protein found in RBCs responsible for carrying oxygen form the lungs to tissues and transporting carbon dioxide back to lungs for exhalation

diagnosing anemia

monitoring oxygen delivery

evaluating nutritional status

assessing blood loss

monitoring chronic conditions

clinical importance of hemoglobin monitoring

cyanmethemoglobin method

requires mixing of Drabkin’s reagent (corrosive) to a sample of blood forming a very stable, colored compound —> cyanmethemoglobin

oxidized blood is read in a spectrometer

HgB + cyanide = cyanmethemoglobin

cyanmethemoglobin reaction

cyanmethemoglobin reaction

RBCs are lysed —> Fe2+ of the released Hgb is oxidized to ferric state (Fe3+) to form methemoglobin —> reacts with cyanide of K+ cyanide to form cyanomethemoglobin (read by spectrometer at 540 nm)

concentration of Hgb in sample

concentration of cyanmethemoglobin = ?

improper pipetting

dirty or scratched cuvettes

Drabkin’s solution deterioration

sources of error due to technique

pipetting not enough or too much sample

not wiping pipette tip before adding sample to Drabkin’s reagent

improper pipetting

scatter light when light hits the cuvette

dirty/scratched cuvettes

must be kept in a dark bottle as it deteriorates when exposed to light

Drabkin’s solution deterioration

sources of error due to condition of the patient

lipemic samples

increased WBC levels

presence of HgB C or HgB S

increased protein levels

lipemic samples

samples with high lipids are cloudy —> interfere with light absorption by the sample

interfere with light absorption

high WBC counts …

HgB C and/or HgB S

RBCs that are resistant to cyanide lysing of the RBCs —> decreased HgB levels

increased protein levels

multiple myeloma —> light absorption issues

16-23 g/dL

normal newborn hemoglobin values

10-14 d/gL

normal children hemoglobin values

13.5-17.5 g/dL

normal adult male hemoglobin values

12.5-15.5 g/dL

normal adult female hemoglobin values

cyanmethemoglobin procedure

1. pipet 5 mL of Drabkin’s reagent + 20 uL of WB into test tube, rinsing the pipet 2-3x in the solution

2. cover test tube with parafilm, invert and allow to stand at RT for 10 minutes —> adequate time for cyanomethemoglobin to form

3. turn on the spec and set the wavelength to 540

4. insert blank cuvette into spec and close cover —> blank to read 0 abs 100% T

5. read the absorbance