CHEMISTRY REAGENT KITS
 ------->HAEMOGLOBIN KIT (Cyanmethemoglobin)

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CLINICAL SIGNIFICANCE
Decreased blood levels are associated with various anaemias. Increased levels are observed In patients with polycythemia or dehydration.

PRINCIPLE
Haemoglobin +
KjFefCN)--------->Methaemoglobin
KCN--------------->Cyanmethaemoglobin
The brownish coloured cyanmethaemoglobin Is the product of almost all forms of haemoglobin found In blood except HbS and can be measured at 546 nM.

REAGENTS SUPPLIED
Cat. No. A 565
Composition
1.Drabkin's Reagent           1000 mL
   Potassium Ferricyanide    0.60 mMol/L
   Sodium Cyanide               0.77 mMol/L
   Phosphate Buffer            1.00 mMol/L
Contains preservatives and stabilisers.

2.Cyanmethaemoglobin Standard  10 mL
    Haemoglobin                  15.06 Gms/dL

Equivalent to 15.06 Gms/dL in assay condition.(Contains buffers, stabilisers and preservatives).

Preparation of Working Reagent
All Reagents are ready to use.

STABILITY
Drabkin's Reagent is stable at least 18 months at 20-35°C, away from light. The standard is stable at 2-8°C for 18 months.

SAMPLE
Fresh blood from finger prick is preferred. Anticoagulated whole blood can be used. Common anticoagulants such as Oxalate, EDTA or Heparin have no effect on this assay. (All samples should be handled as potential infective agents as no laboratory methods make conclusive findings for its safety. Therefore, adequate protective laboratory measures should be taken while handling such materials).

SYSTEM PARAMETERS

Reaction End-point
Temperature 25°C to 35°C
Wavelength 546 nM (530-550 nM)
Standard Concentration
(assay condition) 15.06Gms/dL
Absorbance Range 0-2A0
Cuvette Path Length   1 cM
Reagent Volume 2.50 mL 5.00 mL
Sample Volume 0.01 mL 0.02 mL
Incubation

5 Minutes

Linearity 20 Gms/dL
Max. limit of Blank Reagent

0.070

Final Colour Stability 30 Mins.

MANUAL METHOD
1
   Pipette into 2 Test Tubes Blank mL Test mL
   Drabkins Reagent No.1 5.00 5.00
   Sample (Fresh Blood) - 0.02

2.Mix well. Incubate at RT for 5 minutes.
3.Read at 546 nM (530-550 nM) or GREEN filter against Blank. The final colour is stable for    30 minutes.

MICRO METHOD FOR DISCRETE ANALYSERS
Drabkin's Reagent................................     2.5 mL
Sample....................................................     0.01 mL
Mix well. Incubate at RT for 5 minutes. Read O.D. at 546 nM (530 - 550 nM) against Blank. The final colour is stable for 30 minutes. Multiply results by factor or compute as given under result columns.

CALIBRATION GRAPH

1

   Pipette into 3 Test tubes Tube1 mL Tube1 mL Tube1 mL
   Drabkin's Reagent 5.00 2.5 0.0
   Cyanmethemoglobin Standard 0.0 2.5 5.0
   Read O.D. at 546nM    (530- 550nM) or GREEN filter.
   Corresponding Hb concentration
0.0 7.53 15.06

2. Draw a line, expressing Gms/dL on the X-axis and optical densities on the Y-axis of a      graph. Extrapolate the line within the limit of the dynamic range of this methodology      (20Gms/dL).

NOTE: Programme the analyser using system parameters. A specific programme data sheet may be provided for each analyser upon request.
_________________________________________________________

RESULTS
Haemoglobin In Gms/dL = OD Test x Factor
               15.06
Factor=-------------
              OD STD

             O.D.Test
OR = ---------------- x 0.06 x 251
              O.D.Std

             O.D.Test
OR = ---------------- x 0.06 x 15.06
             O.D.Std

Compare the result from the Calibration graph.

EXPECTED VALUES
Since there is no acceptable Indian values established beyond arbitration, the values given here are of European references.
Men 13.5  to  18.0   Gms/dL
Women 12.0   to   16.0 Gms/dL

Infants at

   
birth 13.6  to  19.6 Gms/dL

Children below 10 years may show slightly lower values. As with all diagnostic methods, the final diagnosis should not be made on the result of a single test as well as laboratory diagnosis must be confirmed with clinical manifestations.

LIMITATIONS
This reagent system is highly interfered by PAP reagent system used in GOD - POD, Cholesterol Oxidase, GPO-PAP, Uricase - PAP etc. For values higher than 20 Gms/dL dilute blood sample 1 to 2 with 0.9% Sodium Chloride an perform the assay. Multiply the result by dilution factor i.e. by 2 for 1:1 dilution.