Hemoglobin, Hematocrit, ESR determination, RBC indices

Hemoglobin

• is responsible for the carrying of oxygen from the lungs which will be delivered to the tissues

• is responsible for the red color of the blood

• is a conjugated protein

respiratory function

Primary function of Hemoglobin in the body

Globin

The entire molecule of hemoglobin is composed of a protein component called?

Conjugated proteins

• Soluble in plasma

• It can mix in the plasma without destroying that substance in the plasma.

• Heme (iron)

• Globin (Protein)

Hemoglobin molecule is composed of:

Heme (iron)

• The specific component of hemoglobin molecule that is responsible for the red color of the blood.

• has to be maintained in its Fe2+ (ferrous/reduced) state all the time in order for the hemoglobin molecule to be fully functional and to perform its respiratory function in the body.

Unconjugated proteins

• Soluble in plasma

• It can mix in the plasma without destroying that substance in the plasma.

Because the body cannot tolerate for the hemoglobin molecule to be

Why is hemoglobin always inside the RBC?

TRUE

TRUE OR FALSE: hemoglobin molecule is a small type of protein, it can easily pass through the filtration rate of the glomerulus. Also, since it is high in molecular weight, constant filtration of hemoglobin in the glomerulus of the kidney will eventually damage the kidneys of the patient.

1. Haptoglobin

2. Hemopexin

3. Albumin

Body defense mechanisms that neutralize the liberated hemoglobin form RBC:

Haptoglobin

• A normal, large protein found in the plasma; the transport protein of hemoglobin molecule

Haptoglobin:Hemoglobin complex

When there is hemolysis, as the hemoglobin is released to the plasma (because RBCs are abnormally destroyed), the haptoglobin will immediately bind to the liberated hemoglobin, forming a complex called?

1:1

What is the ration of haptoglobin:hemoglobin?

Irreversible

The degradation of haptoglobin:hemoglobin complex in the liver is

Hemolytic episode

In detecting for the levels of haptoglobin in the plasma (blood sample) and if the level of the haptoglobin is severely decreased or it reaches zero level already, that is a strong indication of?

Hemopexin

Transport protein of heme normally found in plasma in large size (macromolecule)

Globin

In pronounced hemolysis, haptoglobin in plasma is not enough to neutralize the increasing hemoglobin, eventually hemoglobin will split off. What component of Hemoglobin is reused?

Iron toxicity

When there is too much accumulation of heme in the plasma, that will eventually lead to?

Hemopexin:Heme complex

Once heme is released from the hemoglobin molecule, the hemopexin will immediately bind to it, and another complex in the plasma named?

Reversible

Hemopexin will transport the entire complex to the LIVER, and it will be degraded in the liver, but the degradation of the complex in the liver is?

TRUE

TRUE OR FALSE: After the hemopexin transports and delivers the entire complex to the liver, only the heme portion of the complex gets degraded in the liver.

FALSE

TRUE OR FALSE: the hemopexin levels in the plasma should be used to indicate hemolysis or hemolytic episode.

Albumin

When hemopexin is depleted and it takes time for it to go back to the plasma and bind to free hemoglobin, what protein temporarily attaches to heme?

Methemoglobin (Fairley’s pigment)

What compound is formed when albumin in the plasma attach to the remaining heme?

TRUE

TRUE OR FALSE: The attachment of the albumin to heme is temporary, and the formation of the methemalbumin is also temporary to prevent heme from going to the kidneys and causing toxicity there.

Schumm test

• A laboratory test that determines the presence of methemalbumin in the patient’s blood.

• It differentiates intravascular hemolysis (inside the blood vessel) from extravascular hemolysis (outside the blood vessel).

brown discoloration

What is the positive result of Schumm test/

Protect the kidneys from being damaged when there is hemolysis or hemolytic episodes

What is the main objective of haptoglobin, hemopexin, and albumin?

TRUE

TRUE OR FALSE: The oxygen carrying capacity of the blood is directly proportional to the hemoglobin content

• 4 Globin

• 4 Protoporphyrin IX

• 4 Iron (Ferrous / Fe2+)

• 1 2,3-Diphosphoglycerate (2,3-DPG)

Composition of Hemoglobin:

2 α and 2 β chains

Globin contains 2 pairs of polypeptide chains

10th amino acid sequence

The δ chain is differentiated from the β chain in the?

39th amino acid sequence

The γ chain is differentiated from the β chain in the?

Protoporphyrin IX

is synthesized partly in the cytoplasm of the nucleated RBC during maturation and partly inside the mitochondria of the nucleated RBC

2,3-DPG (Diphosphoglycerate)

• is synthesized in the anaerobic glycolytic pathway

• dictate the oxygenation state of the hemoglobin

Embden-Meyerhof Pathway

2,3-DPG (Diphosphoglycerate) molecule is synthesized in the anaerobic glycolytic pathway called?

Generation of energy for the RBCs

Primary objective of the Embden-Meyerhof Pathway is for the?

Rapoport-Luebering shun

Specifically, the 2,3-DPG molecule is synthesized in the sub pathway of the Embden-Meyerhof Pathway, called?

Tense “T” Form

• If the 2,3-DPG molecule is present & stays in the center of the hemoglobin molecule

• an oxygen molecule is bound to the hemoglobin molecule

• It is non-oxygenated because it is carrying already an oxygen molecule, so it is no longer capable of binding to another oxygen molecule.

Relaxed “R” Form

• If the 2,3-DPG molecule is expelled from the hemoglobin molecule

• This form of hemoglobin has increased affinity for oxygen binding

• Oxygenated state

• Oxyhemoglobin (HbO2)

• Deoxyhemoglobin (HbCO2 or HHb)

Normal/Functional hemoglobin derivatives:

• Carboxyhemoglobin (HbCO)

• Sulfhemoglobin (HbS)

• Methemoglobin / Hemiglobin (Hi)

Abnormal/Non-functional hemoglobin derivatives:

Oxyhemoglobin

A hemoglobin molecule reversibly bound to oxygen

Deoxyhemoglobin

Oxygen is no longer in the hemoglobin molecule (reduced)

Carboxyhemoglobin (HbCo)

• Hemoglobin molecule bound to carbon monoxide

• is irreversible and unstable

Carbon monoxide (CO)

is the primary interference that affects the affinity of the hemoglobin molecule to the oxygen molecule, because the carbon monoxide is a substance where hemoglobin is greatly attracted to.

it will not be converted back into its normal functional type

Why is carboxyhemoglobin irreversible?

it is immediately cleared out by reticuloendothelial system

Why is carboxyhemoglobin unstable?

Senescent red cells

RBCs which are already 120 days old in the circulation and ready for clearing in the reticuloendothelial system

Culling

The term used to describe the clearing of senescent RBCs in the circulation by the reticuloendothelial system; It is the process of clearing senescent red blood cells into circulation by the reticuloendothelial system

Sulfhemoglobin (HbS)

• Hemoglobin molecule bound to sulfides

• is irreversible and stable

will not be cleared out in the RES until the RBC becomes senescent

why is sulfhemoglobin stable?

0 - 2.2% of total hemoglobin

Normal values of sulfhemoglobin

0 - 2.3% of total hemoglobin

Normal values of carboxyhemoglobin in non-smokers

2.1 - 4.2% of total hemoglobin

Normal values of carboxyhemoglobin in smokers

Methemoglobin/Hemiglobin

• Formed when the reduced form of iron in the hemoglobin molecule is oxidized into its oxidized form (ferric form/Fe3+) (Conversion of Fe2+ à Fe3+)

• is reversible and unstable.

methemoglobin reductase / diaphorase enzyme / NADH-dependent methemoglobin reductase / Cytochrome-B5- reductase

Enzyme that can convert the methemoglobin back to its functional type

1.5 - 2.2%

Normal values of methemoglobin in premature infants

1 - 1.5%

Normal values of methemoglobin in infants up to 1 year old

1%

Normal values of methemoglobin in older children and adult

Inherited Methemoglobinemia

• inherits the gene from the parents, where the gene has DIAPHORASE DEFICIENCY (does not contain the enzyme methemoglobin reductase)

• Responds well to methylene blue therapy.

Methylene blue

is a redox dye (a reduction-oxidation dye) capable of performing reduction and oxidation properties.

Acquired methemoglobinemia

• Triggered by substances that can either be ingested or absorbed

• also responds very well to methylene blue therapy

Inherited hemoglobin M methemoglobinemia

Does not respond to methylene blue therapy

Hemoglobinometry

It is the laboratory test that will determine the levels of hemoglobin or hgb concentration in a given blood sample.

Direct visual colorimetric method

Directly observing the change of color of the blood sample as the different reagents are added to the blood sample

Tallquist Method

• Uses absorbent pads with lithographed color scale representing values ranging from 10-100%

• change of colors in the colored pads of the lithograph paper is directly observed.

Dare’s hemoglobinometer

change of colors in the colored pads of the lithograph paper is directly observed.

Acid hematin methods

Hemoglobin is converted into acid hematin

0.1 N HCl

hemoglobin is converted into acid hematin by what acid reagent?

Alkaline hematin methods

Hemoglobin is converted into alkali hematin

NaOH

Hemoglobin is converted into alkali hematin with what reagent?

Oxyhemoglobin method

A method wherein it only determines the normal or functional types of hemoglobin derivatives (only oxyhemoglobin derivatives)

s 0.007 N NH4OH or 0.1% Na2CO3.

The reagent used in oxyhemoglobin method where it converts a specific hemoglobin derivative to oxyhemoglobin

TRUE

TRUE OR FALSE: 0.007 N NH4OH or 0.1% Na2CO3 should be prepared in copper-free glassware or water should be glass distilled.

Shake’s test

A screening test used to determine the presence of these hemoglobin derivatives in the blood sample of the patient.

Bright red

Shake’s test

• Color of oxyhemoglobin after vigorous shaking

Cherry red

Shake’s test

• Color of carboxyhemoglobin after vigorous shaking

Chocolate brown

Shake’s test

• Color of methemoglobin after vigorous shaking

Mild lavender

Shake’s test

• Color of sulfhemoglobin after vigorous shaking

Cyanmethemoglobin method / Hemiglobin Cyanide method / Ferrihemoglobin Cyanide method (HiCN)

• Routinely used method for hemoglobinometry

• Best method because the pigment (cyanmethemoglobin) that is measured in this particular method is a stable pigment.

• All the hemoglobin derivatives are measured by this method, except for sulfhemoglobin since its formation in the blood is stable

• Potassium Ferricyanide

• Potassium Cyanide

Reagents used to effectively quantitate the hemoglobin concentration

Cyanmethemoglobin

is detected at a specific wavelength of 540 nm

• Sodium (Na) Bicarbonate

• Potassium (K) Ferricyanide

• Potassium (K) Cyanide

• Distilled Water

Original Drabkin’s reagent:

Sodium (Na) Bicarbonate

• Responsible for the hemolysis or lysis of the RBCs in the given blood sample.

• The turnaround time of the test using original Drabkin’s reagent is at 15 minutes

• It is a very poisonous solution so the entire reagent must be kept in a locked cabinet at all times when not in use.

Potassium (K) Ferricyanide

Responsible for the oxidation of the Fe2+ in the normal hemoglobin to Fe3+ (methemoglobin)

Potassium (K) Cyanide

Provides cyanide ions to where the methemoglobin will actually attach to, forming the stable pigment cyanmethemoglobin or hemoglobin cyanide.

Dihydrogen Potassium Phosphate (KH2PO4)

• Responsible for the hemolysis or lysis of the RBCs at a shorter timing. 

• It completely lyses the RBCs in a given blood sample at 3 minutes (shorter turnaround time).

Nonionic Detergent

• Helps dihydrogen potassium phosphate in the lysis of the RBCs at a much shorter time.

• It also decreases the amount of turbidity resulting from abnormal proteins.

Photosensitive

The Drabkin’s reagent must be stored in a brown reagent bottle because of what characteristic?

540 nm wavelength

To determine the clarity (appearance) of the reagent, measure the absorbance of the questioned Drabkin’s reagent in a spectrophotometer at?

Turbidity

• Main source of error for hemoglobin concentration determination in Cyanmethemoglobin method / Hemiglobin Cyanide method / Ferrihemoglobin Cyanide method (HiCN)

,000 to 11,000 cells per cubic millimeter

Normal WBC count in the body ranges from?

Solution is centrifuged and transmittance/ absorbance of the supernatant is determined.

What is the remedy of high WBC count?

Lipemic blood

Appearance of the blood is whitish in color because of cholesterol, or if the patient has taken a lot of fatty foods.

the blood sample is first submitted to spinning, and after spinning the blood sample which is lipemic in appearance, the plasma of that spun blood sample is used in making 1:251 dilution (0.02 mL of the patient’s plasma + 5 mL of Drabkin’s reagent).

Remedy for lipemic blood

Hemoglobin S (Hb S/ Sickling Hemoglobin) & Hemoglobin C (Hb C)

Presence of sickling hemoglobin in the RBCs will resist hemolysis, even if the reagent is already added

Add 0.1 g potassium carbonate to the 1:251 dilution (only for Original Drabkin’s Reagent)

Remedy for abnormal proteins/globulins

Copper sulfate method (CuSO4)

• Based from the specific gravity or density of the blood

• The specific gravity or density of the blood is compared to the specific gravity or density of the copper sulfate solution.

• The specific gravity of the copper sulfate in this method is directly proportional to the specific gravity or density of the blood

hemoglobin concentration for screening blood donors

Copper sulfate method (CuSO4) is commonly used to determine?