ACCURATE DETERMINATION OF HEMOGLOBIN Flashcards by Kyra Catedral

→ Ordered by physicians to diagnose and monitor the course of ANEMIA

HEMOGLOBIN

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T or F

HGB can be performed separately or as part of the routine COMPLETE BLOOD COUNT (CBC)

True

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Hematologic analyzers to date include hemoglobin determination as a STANDARD TEST included in____

CBC

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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)

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HGB Transports OXYGEN and CARBON DIOXIDE

When fully saturated, each gram of hemoglobin holds_____ of oxygen.

1.34 mL

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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

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Heme structure

Protoporphyrin IX
Iron atom in the ferrous form (Fe2+)

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Heme consists of a ring of (3) atoms called ________with a divalent_____

carbon, hydrogen, and nitrogen

PROTOPORPHYRIN IX

FERROUS IRON (Fe2+)

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When ferrous iron → oxidized to ferric
state (Fe3+) =________?

CAN NO LONGER BIND TO
OXYGEN

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Each heme is precisely located in a______ of one of the polypeptide chains

pocket or fold

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Hemoglobin is made up of 4…

PROSTHETIC HEME GROUPS

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If iron is lacking (decrease dietary intake or increase loss from the blood) → Hemoglobin is not formed sufficiently → thus leading to…

ANEMIA

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Ferrous (Fe2+) reversibly bound to oxygen

Relaxed form

OXYHEMOGLOBIN

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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

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Ferrous (Fe2+) NOT bound to oxygen
TENSE FORM (with 2,3-BPG)

DEOXYHEMOGLOBIN

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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

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Otherwise, when it is oxidized to ferric state,_______ is formed, and the molecule loses its capacity to efficiently transport oxygen or carbon dioxide.

methemoglobin

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Intrauterine
Early embryogenesis (product of yolk sac erythroblasts)

Globin chain

Hemoglobin

Gower-1 — ez
Gower-2 — ae
Portland — zg

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Intrauterine

Begins in early embryogenesis; peaks during third trimester and begins to decline just before birth

Fetal hemoglobin— AG

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Birth

HbF — AG (60-90%)
HbA — AB (10-40%)

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Two Years through Adulthood

HbA1 —>95%
HbA2 —<3.5%
HbF —1%-2%

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A.k.a dysfunction hemoglobins
Unable to transport oxygen.

DYSHEMOGLOBIN

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Form and may accumulate to toxic levels, after exposure to certain triggers.

DYSHEMOGLOBIN

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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

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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

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

1. SPECIFIC GRAVITY METHOD (COPPER SULFATE METHOD) 2. ACID HEMATIN METHOD (SAHLI-HELLIGE METHOD) 3. 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 1. Prepare the materials. 2. Add _____up to the___ mark on the_____. 3. Pipette blood up to the____ mark (equivalent to_______). Wipe the external surface of the pipette to remove any excess blood. 4. 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. 5. Allow to stand undisturbed for____ mins. 6. 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. 7. Remove the glass rod and take the reading directly by noting the____ of the solution. 8. 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. 4. Allow mixture to stand at_____ temperature for at least 10 minutes (or 3 minutes, depending on the reagent used) 5. Transfer the contents of the tubes into cuvettes 6. Run the "blank" into the spectrophotometer and read absorbance. 7. Place the standard into the cuvette and read absorbance. 8. Run the patient's sample and read absorbance. 9. Calculate hemoglobin concentration using the formula 10. Discard all specimens and contaminated materials into biohazard container. 11. 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

ACCURATE DETERMINATION OF HEMOGLOBIN Flashcards

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