CURTISS

CHEM-INDEX INC. Hialeah, FL 33012

DIRECT HEMOGLOBIN REAGENT

FOR THE COLORIMETRIC DETERMINATION OF HEMOGLOBIN IN BLOOD

3. - ACCURATE DISPENSING DEVICE FOR MEASURING

REAGENT AND SAMPLE SUCH AS SAHLI PIPETTES AND
SEROLOGICAL PIPETTES.

THE CYANMETHEMOGLOBIN STANDARD IS A STABILIZED

PREPARATION OF HUMAN HEMOGLOBIN WITH AN ACCURATELY ASSIGNED VALUE AND IS USED AS A CALIBRATOR FOR
THE HEMOGLOBIN PROCEDURE.

MIX AND ALLOW TO STAND 5 MINUTES AT ROOM

TEMPERATURE
------------------------------------------------------------------------------------------ZERO THE INSTRUMENT WITH THE BLANK CUVETTE AT 540
nm. READ AND RECORD THE ABSORBANCE OF THE STANDARD AND UNKNOWN CUVETTES.

PROCEDURE:
------------------------------------------------------------------------------------------CUVETTES
BLANK
STANDARD UNKNOWN
------------------------------------------------------------------------------------------DRABKINS REAGENT (mL) 5.0
5.0
STANDARD (mL)
3.0
0.02
UNKNOWN (.L)

Figure 1. Typical Calibration Curves

Absorbance (540 nm)

SUMMARY AND EXPLANATION:

THE CYANMETHEMOGLOBIN PROCEDURE HAS BECOME
INTERNATIONALLY ADOPTED AS THE REFERENCE METHOD
FOR HEMOGLOBIN ASSAY. THE PRESENT METHOD IS BASED
ON THE ORIGINAL PROCEDURE DESCRIBED BY DRABKIN IN
WHICH CYANIDE AND FERRICYANIDE IN AN ALKALINE MEDIUM CONVERT HEMOGLOBIN TO THE HIGHLY COLORED CYANMETHEMOGLOBIN DERIVATIVE, WHICH MAY BE MEASURED
SPECTROPHOTOMETRICALLY AT 540 nm. THE METHOD IS
ESSENTIALLY A MEASURE OF TOTAL HEMOGLOBIN BECAUSE
REDUCED HEMOGLOBIN, OXYHEMOGLOBIN, METHEMOGLOBIN AND CARBOXYHEMOGLOBIN REACT TO GIVE THE CYANMETHEMOGLOBIN DERIVATIVE.

0.7
0.6
0.5
0.4
m
1c

0.3
0.2
0.1
5.0

REAGENTS
FOR IN VITRO DIAGNOSTIC USE
1.- DRABKINS REAGENT POWDER.- SODIUM BICARBONATE,
100 PARTS, POTASSIUM FERRICYANIDE, 20 PARTS, AND
POTASSIUM CYANIDE, 5 PARTS STORE IN DARK AT ROOM
TEMPERATURE (18- 26C)
DRABKINS SOLUTION IS PREPARED BY RECONSTITUTING DRABKINS REAGENT POWDER WITH 2000 ML
DEIONIZED WATER. ADD 1.0 ML 30% BRIJ-35 SOLUTION,
AND MIX WELL. FILTER IF INSOLUBLE PARTICLES
REMAIN. STORE IN AMBER BOTTLE AT ROOM TEMPERATURE (18- 26C). STABLE FOR AT LEAST 6 MONTHS.
DANGER: POISON. MAY BE FATAL IF SWALLOWED.
DRABKINS REAGENT AND DRABKINS SOLUTION
CONTAIN CYANIDE.
2. BRIJ-35 SOLUTION.
BRIG-35, 30 G/DL. STORE AT ROOM TEMPERATURE (186C) DISCARD IF TURBIDITY DEVELOPS. IF SOLUTION
SOLIDIFIES, WARM TO 37C TO LIQUEFY.
WARNING: CAUSES IRRITATION. AVOID CONTACT WITH
EYES, SKIN AND CLOTHING. AVOID BREATHING VAPOR.
WASH THOROUGHLY AFTER HANDLING.
3. HEMOGLOBIN STANDARD. CONTAINS 80 g/dL CYANMETHEMOGLOBIN CORRESPONDING TO 20 g/dL HEMOGLOBIN
WHEN USED ACCORDING TO THE HEMOGLOBIN
PROCEDURE. THE STANDARD IS PREPARED BY ADDITION
OF HUMAN HEMOGLOBIN TO CYANMETHEMOGLOBIN
REAGENT. STORE REFRIGERATED 2- 8 C PROTECT
FROM LIGHT. IF THE SOLUTION APPEARS TURBID,
DISCARD IT AND DO NOT USE. PERMIT THE STANDARD TO
REACH ROOM TEMPERATURE BEFORE USE.
CAUTION: REAGENTS CONTAINS CYANIDE. DO NOT MIX
WITH ACID. DO NOT PIPETTE BY MOUTH. AVOID CONTACT
WITH SKIN. IF INGESTED, PERFORM GASTRIC LAVAGE AND
CALL A PHYSICIAN. DISCARD BY FLUSHING WITH COPIOUS
AMOUNTS OF WATER.
SPECIMEN COLLECTION AND PREPARATION: THIS METHOD
REQUIRES WHOLE BLOOD CONTAINING HEPARIN, OXALATE,
CITRATE OR EDTA AS ANTI-COAGULANT. THE HEMOGLOBIN
CONTENT OF BLOOD SPECIMENS COLLECTED WITH AN ANTICOAGULANT IS STABLE ABOUT ONE WEEK AT ROOM TEMPERATURE.
MATERIALS REQUIRED BUT NOT SUPPLIED:
l. - COLORIMETER OR SPECTROPHOTOMETER WITH
APPROPRIATE WELL, SUITABLE FOR MEASUREMENT AT
540 nm.
2. - TIMING DEVICE

CALCULATIONS:
THE FOLLOWING EQUATION IS USED TO DETERMINE
UNKNOWN CONCENTRATION:
UNKNOWN ABSORBANCE
STANDARD ABSORBANCE

20

HEMOGLOBIN IN
g/dL

EXAMPLE OF CALCULATIONS:
UNKNOWN ABS = 0.48
0.48
0.61

20

STANDARD ABS = 0.61

=

15.8 g/dL

NOTE: FOR DIRECT READOUT INSTRUMENT, SET READOUT

TO CONCENTRATION OF STANDARD AND READ THE
UNKNOWN CONCENTRATIONS DIRECTLY.
METHOD USING CALIBRATION CURVE:
AN ALTERNATE METHOD FOR STANDARDIZATION OF THE
HEMOGLOBIN PROCEDURE IS THE USE OF A CALIBRATION
CURVE. BY THIS METHOD, A STRAIGHT LINE IS GENERATED
AND THE RESULTS MAY BE READ DIRECTLY FROM A GRAPH.
------------------------------------------------------------------------------------------HG CONCEN. (g/dL) BLANK
5
10
15
20
------------------------------------------------------------------------------------------STANDARD (mL)
1.0
2.0
3.0
4.0
DRABKINS (mL)
4.0
3.0
2.0
1.0
------------------------------------------------------------------------------------------MIX, AND ZEROING THE INSTRUMENT WITH THE BLANK
CUVETTE AT 0.00 ABSORBANCE, SET AT 540 nm. AND READ
THE ABSORBANCES OF THE REST OF THE CUVETTES AND
RECORD THEM. PLOT THE ABSORBANCE OF EACH DILUTION
VERSUS THE ASSIGNED HEMOGLOBIN CONCENTRATION.
RESULTS:
RESULTS ARE OBTAINED DIRECTLY FROM THE CALIBRATION
CURVE.
EXAMPLE: A SPECIMEN ASSAYED BY THIS PROCEDURE
YIELDED AN ABSORBANCE OF 0.340 USING 1 cm CUVETTE.
FROM TYPICAL CALIBRATION CURVES (Figure 1) A BLOOD
HEMOGLOBIN VALUE OF 15.0 g/dL IS OBTAINED.

t
ve
cu

10.0

15.0

20.0

Blood Hemoglobin (g/dL)

NOTE: TYPICAL CALIBRATION CURVES (Figure 1) ARE PRESENTED FOR ILLUSTRATIVE PURPOSES AND MUST NOT BE
USED TO CALCULATE TEST RESULTS.
EXPECTED VALUES:
Normal Range
HEMOGLOBIN
(g/dL)
------------------------------------------------------------------------------------------ADULT MALES
13 - 18
ADULT FEMALES
11 - 16
NEWBORNS
14 - 23
------------------------------------------------------------------------------------------A NUMBER OF FACTORS, SUCH AS AGE, RACE, EXERCISE,
SEASON AND ALTITUDE, ARE REPORTED TO INFLUENCE THE
VALUES OF NORMAL RANGES AS SHOWN ABOVE.
INCREASED HEMOGLOBIN VALUES ARE OBSERVED IN CASES
OF PRIMARY AND SECONDARY POLYCYTHEMIA, SEVERE
DEHYDRATION, CONGESTIVE HEART FAILURE AND IN BLOOD
FROM PEOPLE LIVING AT HIGH ALTITUDES. LOW VALUES ARE
ENCOUNTERED IN A VARIETY OF ANEMIAS. A COMPREHENSIVE REVIEW BY YOUNG REVEALS THAT NUMEROUS DRUGS
EXERT AN IN VIVO EFFECT TO GIVE DECREASED WHOLE
BLOOD HEMOGLOBIN VALUES.
QUALITY CONTROL:
THE REGULAR USE OF CONTROL BLOOD IS RECOMMENDED
AS PART OF A QUALITY CONTROL PROGRAM.
LIMITATIONS OF THE METHOD:
THE EXTREMELY LOW CONCENTRATIONS OF HEMOGLOBIN
IN PLASMA PRECLUDE THE USE OF THIS SPECIMEN IN THIS
PROCEDURE. BLOOD COLLECTED WITHOUT AN ANTICOAGULANT (SUCH AS THAT OBTAINED FROM A FINGER PUNCTURE)
MUST BE EMPLOYED EXPEDITIOUSLY IN ORDER TO AVOID
CLOTTING OF THE SPECIMEN.
IF CONDENSATION APPEARS ON THE INNER WALL OF THE
BOTTLE, MIX WELL BEFORE USE.
PERFORMANCE CHARACTERISTIC OF THE TEST
A.cCuracy
LiNEAR REGRESSION ANALYSIS OF 40 SAMPLES OF WHOLE
BLOOD, WITH HEMOGLOBIN CONCENTRATION RANGING
FROM 6.8 AND 17.8 g/dL WAS PERFORMED COMPARING THE
PRESENT METHOD WITH OTHER COMMERCIALLY AVAILABLE
REAGENT, BASED ALSO ON THE CYANMETHEMOGLOBIN
METHOD, WITH THE FOLLOWING RESULTS:
y (curtiss) = 1.002 . + 0.1
r (CORRELATION COEFFICIENT) = 0.998

PRECISION
BETWEEN RUN PRECISION WAS DETERMINED BY ASSAYIN
TWO WHOLE BLOOD SAMPLES, 7 TIMES DAILY OVER A 3 DAY
PERIOD (N=21):

MEAN (g/dL)
STANDARD DEVIATION (g/dL)
COEFFICIENT OF VARIATION (%)

SAMPLE #1
11.2
0.31
2.77

SAMPLE #2
15.8
0.33
2.1

SENSITIVITY
UNDER THE CONDITIONS DESCRIBED ON PROCEDURES A
CHANGE OF 0.001 ABSORBANCE UNITS, WHEN MEASURED IN
1 CM LIGHT-PATH CUVET, IS EQUAVALENT TO APPROXIMATELY 0.27 g/dL.

REFERENCES
1. Stadie, W. C., J. Biol. Chem. 41, 237, 1920.
2. Van Kampen, E.J. and Zijlstra. W.G. Adv., Clin. Chem. 8,
141,1965
3. ICSH recommendations for hemoglobinometry in humen blood.
Br. J. Haematol. 13 (Supl.), 71,1967.
4. ICSH recommendations for reference method for hemaglobinometry in human blood (ICSH Standard EP 6/2: 1977) and
Specifications for international hemiglobincyanide reference
preparation (ICSH Standard EP 6/3: 1977). J. Clin. Pathol. 31,
139, 1978.
5. Rice, E.W., Koepke, J.A. and Evatt, B.C., In Selected Methods of
Clinical Chemistry, Vol. 9, pp. 265-266 AACC. Washington, D.C.,
1982.
6. Young, D.S., Pestaner, L.C., and Gibberman, V. Clin. Chem. 21,
1D (1975).

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implied, except that the material from which its products are made
are of standard quality. If any materials are defective , CURTISS
will provide a replacement product. Buyer assumes all risk and
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merchantability of this product, or of the fitness of the product for any
purpose. CURTISS shall not be liable for any damages, including special or consequential damage, or expense arising directly
or indirectly from the use of this product.

226-6LS
10/26/04