METHOD HISTORY

Previous methods used for the determination of blood hemoglobin were based on estimations of oxygen or carbon monoxide capacity or iron content. Of all methods, only the cyanmethemoglobin has gained popular acceptance. The original cyanmethemoglobin technique was proposed by Stadie in 1920.1 This method used separate alkaline ferricyanide and cyanide reagents. A single reagent was introduced by Drabkin and Austin in 1935. In 1958 the National Research Council (NRC) recommended adoption of the cyanmethemoglobin procedure based on field trials conducted by the Army Medical Department.3,4 In 1966 the International Committee on Standardization in Hematology approved the proposal that all clinical laboratories should adopt this method exclusively.(Obelis S.A 2010)

HISTORY

Originally a colorimetric cyanmethemoglobin method was proposed by Stadie in 1920 [184],

were total hemoglobin at alkaline pH is rapidly converted to the cyanoderivative, whose

absorbance was then measured at 540 nm. This method was then simplified by Drabkin and

Austin in 1935 by combining the separate reactants alkaline ferricyanide and cyanide into a

single reagent. Basically Drabkin’s assay is based on the oxidation of hemoglobin and its

derivatives (except sulfhemoglobin, a pigment that normally occurs in only minute

concentrations in blood) to methemoglobin in the presence of alkaline potassium ferricyanide.

Methemoglobin then reacts with potassium cyanide to form cyanmethemoglobin, which has

maximum absorption at 540 nm. The color intensity measured at 540 nm is proportional to the

total hemoglobin concentration. The color is suitable for measurement in filter as well as in

narrow-band spectrophotometers because its absorption band at a wavelength of 540 nm is broad

and relatively flat, thereby permitting measurements using both wide and narrow-bandwitdth

instruments(530 to 550nm). The addition of specific surfactant also minimizes the turbidity

occasionally caused by the presence of RBC stromae. Drabkin’s reagent still remains the

benchmark against which all other methods should be tested. (Lippi, et al. 2012)

Cyanmethemoglobin refers to a relatively non-toxic cyanide derivative of methemoglobin formed when amyl nitrite and sodium nitrite are used as antidotes to cyanide poisoning. Methemoglobin is an oxidized form of hemoglobin, containing ferric rather than ferrous iron that cannot transport oxygen. (Mifflin 2004)

a compound formed by combination of hydrocyanic acid with methemoglobin. It is formed when methylene blue is administered as an antidote in cyanide poisoning. In laboratory methods, hemoglobin is converted to cyanmethemoglobin and measured spectrophotometrically. (Elsevier 2007)

PRINCIPLEBlood is diluted in an alkaline Drabkin solution of potassium ferricyanide, potassium cyanide, sodium bicarbonate, and a surfactant. The hemoglobin is oxidized to methemoglobin (Fe3+) by the potassium ferricyanide, K3Fe(CN)6. The potassium cyanide (KCN) then converts the methemoglobin to cyanmethemoglobin. The absorbance of the cyanmethemoglobin at 540 nm is directly proportional to the hemoglobin concentration. Sulfhemoglobin is not converted to cyanmethemoglobin; it cannot be measured by this method. Sulfhemoglobin fractions of more than 0.05 g/dL are seldom encountered in clinical practice. (Rodak 2012)

MethemoglobinMethemoglobin is an abnormal Hb in which iron is oxidized from its ferrous to ferric state.

Therefore, it is incapable of carrying oxygen. Normally it is present in low concentrations, but its

formation increases in the presence of certain chemicals or drugs. The formation of

methemoglobin is also reversible.( Venkatesh 2004)

Cyanmethemoglobin (hemoglobin-cyanide)

This is formed by the action of a chemical called cyanide (for example. Potassium

cyanide, KCN). The combination is reversible. Hemiglobincyanide is the methemoglobin

bonded to cyanide ions. Note: To measure accurately the total Hb in the blood, it is

essential to prepare a stable derivative that will contain all the a Hb forms (complexes)

that are present in the blood. All forms of circulating hemoglobin are readily converted to

hemoglobin-cyanide (cyanmethemoglobin), except for sulfhemoglobin which is normally

not present in the blood. Therefore, the cyanmethemoglobin method is the most

accurate method for the determination of hemoglobin.(Venkatesh 2004)

SOURCES OF ERROR AND COMMENTS (RODAK 2012)

1. cyanmethemoglobin reagent is sensitive to light. It should be stored in a brown bottle or

in a dark place.

2. A high leukocyte count (greater than 20 x 10^9/L) or a high platelet count (greater than

700 x 10^9/L can cause turbidity and a falsely high result. In this case, the solution can be

centrifuged and the supernatant measured.

3. Lipemia also can interfere. A false result can be corrected by adding 0.01 mL of the

patient’s plasma to 5 mL of the cyanmethemoglobin reagent and using this solution as the

reagent blank.

4. Cells containing Hb S and Hb C may be resistant to hemolysis, causing turbidity; this can

be corrected by making a 1 : 2 dilution with distilled water (1 part diluted sample plus 1

part water) and multiplying the results from the standard curve by 2.

5. Abnormal globulins, such as those found in patients with multiple myeloma or

Waldenstrom macroglobulinemia, may precipitate. If this occurs, add 0.1 g of potassium

carbonate to the cyanmethemoglobin reagent. Commercially available

cyanmethemoglobin reagent has been modified to contain KH2PO4 salt, so this problem

is not likely to occur.

6. Carboxyhemoglobin takes 1 hour to convert to cyanmethemoglobin and theoretically

could cause erroneous results in samples from heavy smokers. The degree of error is

probably not clinically significant, however

7. Because hemoglobin reagent contains cyanide, it must be used cautiously; a minimum of

4 L of reagent is lethal. Acid free sinks should be used for disposal of reagent and samles,

because acidificatiob of cyanide releases hydrogen cyanide gas. Copious amounts of

water should be usedaa to flush the sink after disposal.

A common source of interference in the cyanmethemoglobin method is: very high WBC countTurbidity in cyanmethemoglobin reagent- patient specimen will result in falsely elevated hemoglobin values. Substance that causes turbidity will influence absorbance measurements of the cyanmethemoglobin. These includes lipids, abnormal plasma proteins or erythrocyte stroma.(LLC 2001)

Interferences (Obelis S.A 2010)

1. Substances that cause turbidity will falsely elevate the hemoglobin value. These include lipids,

abnormal plasma proteins (macroglobulinemia) or

erythrocyte stroma.

2. A review by Young et al 10 reveals the numerous drugs that exert an in vitro effect to decrease

blood hemoglobin values.

Limitations (Obelis S.A 2010)

1. This procedure measures hemoglobin and its derivatives except

sulfhemoglobin.

2. Specimens with values above 20.0 g/dl must be re-run using one-half the

sample volume. Multiply final results by two.

PRECAUTIONS

Universal and Standard precautions must be observed when performing hemoglobin

measurements. In addition, because most hemoglobin reagents contain hazardous chemicals such

as cyanide or azide, care must be taken when performing the test and handling the reagents.

Precautions include wearing gloves, working in a well-ventilated area, properly disposing of

used reagents, wiping up all spills, and handwashing after completion of the procedure.

(Barbara H.Estridge, et al. (2000)

QUALITY ASSURANCE

The use of Drabkin’s reagent to form the stable compound cyanmethemoglobin was an important

advancement in hematology because it made reliable hemoglobin standards available for the first time. A

hemoglobin solution made with Drabkin’s is stable for at least six months and provides laboratories a

reliable standard to use for standardizing hemoglobin assays. (Barbara H.Estridge, et al. (2000)

Spectrophotometers and hemoglobin analyzers used for hemoglobin assays must be calibrated at regular

intervals as specified by the manufacturer. Appropriate control solutions must be run at least daily or

when patient samples are run, control results must be recorded, and the records must be maintained. Each

instrument used will have its own particular checks that must be performed and documented. (Barbara H.Estridge, et al. (2000)

(Wiggers, 2003) Three advantages of the cyanmethemoglobin method are:

1. measures all forms of hemoglobin except sulfhemoglobin

2. can be easily standardized

3. cyanmethemoglobin reagent (also called Drabkin's solution) is very stable