ACCURATE DETERMINATION OF HEMOGLOBIN Flashcards
→ Ordered by physicians to diagnose and monitor the course of ANEMIA
HEMOGLOBIN
T or F
HGB can be performed separately or as part of the routine COMPLETE BLOOD COUNT (CBC)
True
Hematologic analyzers to date include hemoglobin determination as a STANDARD TEST included in____
CBC
FUNCTIONS OF HEMOGLOBIN
Main component of RED BLOOD CELLS
Transports OXYGEN and CARBON DIOXIDE
Acid-base balance
Binding, inactivation, and transport of nitric oxide (NO)
HGB Transports OXYGEN and CARBON DIOXIDE
When fully saturated, each gram of hemoglobin holds_____ of oxygen.
1.34 mL
STRUCTURE OF HEMOGLOBIN
Hemoglobin molecule is composed of two different pairs of polypeptide chains called_______ and four________, with one heme group embedded in each of the four polypeptide chains
GLOBIN
HEME GROUPS
Heme structure
Protoporphyrin IX
Iron atom in the ferrous form (Fe2+)
Heme consists of a ring of (3) atoms called ________with a divalent_____
carbon, hydrogen, and nitrogen
PROTOPORPHYRIN IX
FERROUS IRON (Fe2+)
When ferrous iron → oxidized to ferric
state (Fe3+) =________?
CAN NO LONGER BIND TO
OXYGEN
Each heme is precisely located in a______ of one of the polypeptide chains
pocket or fold
Hemoglobin is made up of 4…
PROSTHETIC HEME GROUPS
If iron is lacking (decrease dietary intake or increase loss from the blood) → Hemoglobin is not formed sufficiently → thus leading to…
ANEMIA
Ferrous (Fe2+) reversibly bound to oxygen
Relaxed form
OXYHEMOGLOBIN
oxygen is taken up at the iron atom until each iron is bound with one oxygen molecule → one hemoglobin molecule is capable of binding with four oxygen molecules
- when such happens, the hemoglobin
is called
OXYHEMOGLOBIN
Ferrous (Fe2+) NOT bound to oxygen
TENSE FORM (with 2,3-BPG)
DEOXYHEMOGLOBIN
When oxygen Is bound to iron, there is no true______ that occurs.
The hemoglobin molecule is simply_____.
Iron must be maintained in its reduced state.
oxidation-reduction
oxygenated
Otherwise, when it is oxidized to ferric state,_______ is formed, and the molecule loses its capacity to efficiently transport oxygen or carbon dioxide.
methemoglobin
Intrauterine
Early embryogenesis (product of yolk sac erythroblasts)
Globin chain
Hemoglobin
Gower-1 — ez
Gower-2 — ae
Portland — zg
Intrauterine
Begins in early embryogenesis; peaks during third trimester and begins to decline just before birth
Fetal hemoglobin— AG
Birth
HbF — AG (60-90%)
HbA — AB (10-40%)
Two Years through Adulthood
HbA1 —>95%
HbA2 —<3.5%
HbF —1%-2%
A.k.a dysfunction hemoglobins
Unable to transport oxygen.
DYSHEMOGLOBIN
Form and may accumulate to toxic levels, after exposure to certain triggers.
DYSHEMOGLOBIN
Dyshemoglobin changes the hemoglobin structure, inhibiting the hemoglobin molecule from binding with oxygen.
Severe impairment = can result to…
If not corrected = will eventually lead to
…
hypoxia or cyanosis
death
T or F
Dyshemoglobin
Most cases are acquired; only a small fraction is hereditary.
True
Normal conc of methemoglobin
Normally 1-2%
Methemoglobin aka
a.k.a Ferrihemoglobin or Hemiglobin
Formed by the reversible oxidation of Ferrous (Fez+) to Ferric (Fe3+); cannot carry O2
Normally occurs (in small amount);
accumulation is prevented by______
Methemoglobin
methemoglobin reduction systems
Color of blood (methemoglobinemia):
chocolate brown
- Normally, there is a small amount of methemoglobin produced during oxygenation and deoxygenation of hemoglobin.
However, it is limited to only___% of the total hemoglobin because of the protective reduction of iron facilitated by__________
1%
NADH-cytochrome b5 reductase 3 (methemoglobin reductase).
Accumulation of methemoglobin in the blood gives it a chocolate brown color and does not revert ack to normal red color after_______ exposure.
oxygen
Most cases of_______ is acquired, resulting from exposure to certain drugs and chemicals such as nitrates, nitrites, quinolones, and chlorates.
methemoglobinemia
Inherited forms of methemoglobinemia may result as an abnormality in globin chain production or as a result of_____
enzyme defect
CARBOXYHEMOGLOBIN (HCO)
________ bound to heme
CO (carbon monoxide)
Carboxyhemoglobin
Carbon monoxide
Has______ times greater affinity to hemoglobin than O2
210
Silent killer; a tasteless, colorless, and odorless gas - can make victimes quickly hypoxic
CO
Carboxyhemoglobin
Binding is reversible
• Carbon monoxide poisoning treatment is use of______ in order to remove carbon monoxide in the blood
hyperbaric oxygen
Color of blood and skin in HbCO poisoning:
cherry red
SULFHEMOGLOBIN (SHb)
Normally_____%
< 1%
Sulfhemoglobin
What gives the greenish color?
Sulfur bound to heme
Mixture of oxidized, partially denatured forms of hemoglobin
Sulfhemoglobin
Irreversible Dyshemoglobin
Sulfhemoglobin
Color of blood in dyshemoglobinemia
mauve-lavender
- During oxidation of hemoglobin, sulfur is incorporated into the heme rings, resulting in a______ hemochrome, imparting a_________color to the blood.
- Further oxidation usually results in the denaturation and precipitation of hemoglobin as______
green
mauve-lavender
Heinz bodies.
-_________ is ineffective for oxygen transport, and patients with elevated levels present with cyanosis.
- Unlike methemoglobin, this cannot be reverted back to normal hemoglobin, and it remains in the cell for all its life.
Sulfhemoglobin
HEMOGLOBIN MEASUREMENT IN THE LABORATORY
- SPECIFIC GRAVITY METHOD (COPPER SULFATE METHOD)
- ACID HEMATIN METHOD (SAHLI-HELLIGE METHOD)
- CYANMETHEMOGLOBIN METHOD (HEMIGLOBINCYANIDE METHOD)
Principle
• Based on the estimation of the Specific gravity of blood
SPECIFIC GRAVITY METHOD (COPPER SULFATE METHOD)
SPECIFIC GRAVITY METHOD (________)
• A blood droplet, allowed to fall into a____, solution with a specific gravity of_____, becomes encased in a sac of______, which prevents dispersion of fluid for_____ seconds
COPPER SULFATE METHOD
CuSO
1.053
copper proteinate
15 seconds
SPECIFIC GRAVITY of 1.053 = ______of
hemoglobin
12.5g/dL
Sample used or collected for CuSo method
Capillary blood
COPPER SULFATE METHOD PROCEDURE
Prepare the CuSO, solution (SG = 1.053) in a wide-mouth container:_____ depth
Perform____
Allow the drop of blood to fall into the CuSO4 solution.
Observe the movement of droplet for at least 15 seconds.
5-inch
skin puncture
CuSo method INTERPRETATION
SG of blood > CuSo sol. = SINK
SG of blood < CuSo sol. = FLOAT
ACID HEMATIN METHOD (_________)
SAHLI-HELLIGE METHOD
ACID HEMATIN METHOD (SAHLI-HELLIGE METHOD)
• Blood + 0.1 N HCI = Hemoglobin →_______ (dark brown colored compound)
• The solution is diluted with distilled water until its color intensity matches with the standard brown colored glass of the______ box.
• The concentration of hemoglobin is read directly on the apparatus.
Acid hematin
comparator box
Acid hematin method
• Sample:
EDTA whole blood
Sahli’s Hemoglobinometer Set
Comparator
Hemoglobin tube
Hemoglobin pipette
Stirrer
SAHLI-HELLIGE METHOD PROCEDURE
- Prepare the materials.
- Add _____up to the___ mark on the_____.
- Pipette blood up to the____ mark (equivalent to_______). Wipe the external surface of the pipette to remove any excess blood.
- Add the blood into the hemoglobinometer containing
HCl. Rinse the contents of the pipette by drawing in and blowing out the acid _____times. Thoroughly mix the blood with the acid. - Allow to stand undisturbed for____ mins.
- Place the hemoglobinometer in the_____ and add______ to the solution dropwise, stirring with the glass rod, until its color matches with that of the comparator glass.
Note: While matching the color, the glass rod must be removed form the solution and held vertically in the tube. - Remove the glass rod and take the reading directly by noting the____ of the solution.
- Report results in______
0.1N HCl ; 2; hemoglobinometer
20th; 0.02mL or 20 uL blood
2-3
10 mins
comparator; distilled water
height
grams per 100 mL of blood (g/dL)
CYANMETHEMOGLOBIN METHOD (________)
HEMIGLOBINCYANIDE METHOD
CYANMETHEMOGLOBIN METHOD (HEMIGLOBINCYANIDE METHOD)
Blood is diluted with_____
_______ oxidizes hemoglobin to_____.
_______ provides cyanide ions to form______ .
Drabkin’s reagent.
Potassium ferricyanide; methemoglobin
Potassium cyanide; cyanmethemoglobin
Cyanmethemoglobin
• The solution is read at______ wavelength and is compared with that of a standard_____ solution.
• Sample:
540mm; HiCN
EDTA whole blood (0.02 mL sample + 5mL reagent)
Drabkin’s Reagent
: oxidizes ferrous iron to ferric state
: donates cyanide ions
: shortens the time needed for the
the complete conversion of Hb to HiCN (___minutes); enhances lysis of erythrocytes and decreased turbidity from protein precipitation.
Potassium ferricyanide
Potassium cyanide
Sodium bicarbonate; 10
Sodium bicarbonate
Replaced by_______ in the modified Drabkin’s reagent: decreases reaction time____ only
Dihydrogen potassium phosphate
3mins
Cyanmethemoglobin
Procedure
1. Prepare all equipment & label tubes as: blank, standard, and unknown.
2. Turn on the spectrophotometer and set the wavelength at____
3. Pipette as follows
a. Dispense 5 mL of _____into blank and unknown test tubes.
b. Dispense 5 mL of____ standard into the standard test tube.
c. Using a micropipette, draw 20 uL of_____.
Wipe excess blood from exterior of pipette with tissue paper. Dispense the blood into the unknown test tube.
- Allow mixture to stand at_____ temperature for at least 10 minutes (or 3 minutes, depending on the reagent used)
- Transfer the contents of the tubes into cuvettes
- Run the “blank” into the spectrophotometer and read absorbance.
- Place the standard into the cuvette and read absorbance.
- Run the patient’s sample and read absorbance.
- Calculate hemoglobin concentration using the formula
- Discard all specimens and contaminated materials into biohazard container.
- Disinfect and clean equipment and return to proper storage.
540 nm
Drabkin’s reagent ; hemoglobin; blood
room
ERRORS INHERENT IN THE SAMPLE
SOURCES OF ERROR
(Falsely elevated Hb)
Turbidity brought by
^ WBC count (>20 x 10^9/L)
^ platelet count (>700 x 10^9/L)
Turbidity brought by
LIPEMIA
Cells containing HbS and HbC
(resistant to lysis = turbidity)
Corrections for
Turbidity brought by
^ WBC count (>20 x 10^9/L)
^ platelet count (>700 x 10^9/L)
Turbidity brought by LIPEMIA
Cells containing HbS and HbC
(resistant to lysis = turbidity)
Centrifuge reagent-sample solution, then the supernatant is measured
Add 0.01mL of the patient’s plasma to 5mL of the Drabkin’s reagent and using this solution as the reagent blank
Make a 1:2 dilution with distilled water (1 part ed sample plus 1 part water) and multiplying the results from the standard curve by 2
ERRORS INHERENT IN THE SAMPLE
Others
Improper venipuncture technique may introduce hemoconcentration.
HbCO (Carboxyhemoglobin) takes about 1 hour to convert to HiCN and theoretically could cause erroneous results in specimens from heavy smokers.
ERRORS INHERENT IN THE METHOD
• Use of the HiCN standard for calibration of the instrument and for the test itself eliminates major source of error.
• The broad absorption band of HiCN in the region of 540 nm makes it convenient to use both in filter-type photometers and in narrow-band spectrophotometers.
• With the exception of SHb, all other varieties of hemoglobin are converted to HiCN.
• Cyanmethemoglobin reagent is sensitive to light.
• It should be stored in a brown bottle or in a dark place.
Cyanmethemoglobin
ERRORS INHERENT IN THE METHOD
• Use of the____ standard for calibration of the instrument and for the test itself eliminates major source of error.
• The broad absorption band of HiCN in the region of 540 nm makes it convenient to use both in filter-type photometers and in narrow-band spectrophotometers.
• With the exception of____, all other varieties of hemoglobin are converted to HiCN.
•______ reagent is sensitive to light.
• It should be stored in a_____
HiCN
SHb
Cyanmethemoglobin
brown bottle or in a dark place.
ERRORS INHERENT IN THE EQUIPMENT
• The accuracy of equipment is not uniform.
• Calibration of pipettes will lesson errors.
• Significant error can be introduced by the use of unmatched cuvettes.
• The wavelength setting, the filters, and the meter readings require checking.
OPERATOR’S ERROR
Reduced by:
Good training
An understanding of the clinical significance of the test
The necessity for a dependable method
Adherence to oral and written instructions
Familiarity with the equipment and with the sources of error
REFERENCE RANGES
MALE: 13.5 - 18 g/dL
FEMALE : 12-16 g/dL
-Haemoglobin pipette -
It’s a slnder special pipette with a single mark ____for taking blood.
20mm (0.02ml)
-Double sided graduated diluting tube-
It is calibrated Hb (2-24g/dL) in_____ colour, and percentage Hb (20-140%) in____ colour.
yellow
red
