1. HEMOGLOBIN DETERMINATION
Cyanmethemoglobin Method

2. INTRODUCTION Hemoglobin (Hb) is the primary constituent of RBC.
The primary function of hemoglobin is to transport oxygen (O2) from the lungs to the tissue cells of the body and to carry carbon dioxide (CO2).

3. INTRODUCTION Hemoglobin has 2 parts as its name indicates:
Heme----Globin
Heme: iron-containing non-protein group (4 in one hemoglobin molecule, each one binds reversibly with one O2)
Globin: a protein consist of four subunits

5. INTRODUCTION Hemoglobin: If bound to O2  iron content appears RED
If bound to CO2  iron content appears BLUE
O2 Bound = Oxygenated
Not Bound to O2 = Deoxygenated

6. INTRODUCTION Each gram of Hemoglobin can carry 1.34 ml of O2
The oxygen combining ability of the blood is in direct proportion to the hemoglobin concentration, rather than the # of RBC

7. MATERIAL Alcohol wipes. Hb pipettes. Cotton Tapes.
Drabkin solution: consist of
Potassium ferricyanide 200mg
Potassium cyanide 50 mg
Potassium dihydroen phosphate 140 mg
Triton-X 1 ml
Distilled water to 1 L
Colorimeter or spectrophotometer.
Test tubes.

8. Cyanmathemoglobin Method
K+ Ferricyanide
Methemoglobin (Fe+³)
Hemoglobin (Fe+²)
Oxidation +
K+ Cyanide
Cyanmethemoglobin (HiCN)

9. CYANMETHEMOGLOBIN METHOD
Compare the color of (Hemiglobincyanide:HiCN) with the standardat 540 nm
Conventionally used in most routine labs

10. METHODOLOGY Add 5 ml of Drabkin reagent to a test tube.
Add 20ul blood to the test tube using Hb pipette or micropipette.
Mix blood with Drabkin’s and wait for 5 to10 minute for the reaction to take place.
Measure the absorbance of the sample against blank (drabkin’s reagent) at 540 nm by spectrophotometer

11. NORMAL HB VALUES ARE: Adult males: 14-18gm Adult females: 12-16 gm
Newborn: gm

12. SIGNIFICANT OF THE TEST:
The measurement of blood hemoglobin is one of the most common clinical laboratory tests.
The hemoglobin test is used to indirectly evaluate the oxygen-carrying capacity of the blood. This makes it an important aid in detecting and evaluating blood loss and diagnosing and treating anemia.

13. SIGNIFICANT OF THE TEST:
Physiological Variation:
HB values are affected by age, sex, pregnancy, and altitude.
Pathological Variation:
Decreased level of HB:
Blood loss.
Bone marrow suppression
Increased level of HB:
Are found in any condition in which the # of RBC rises above normal

14. MECHANICAL SOURCES OF ERROR:
Pipetting error.
Use of dirty or scratched cuvettes.
Use of deteriorated reagent.

15. (Packed Cell Volume of Whole Blood)
HEMATOCRIT
(Packed Cell Volume of Whole Blood)

16. HEMATOCRIT (HCT)
Hct or “ Packed cell volume (PCV) Is a proportion of packed red cells to whole blood in terms of %
Provides information on the amount of red blood cells (RBC) present in the blood.
Decreased levels means anemia from hemorrhage, parasites, nutritional deficiencies or chronic disease process, such as liver disease, cancer, etc.
Increased levels are often seen in dehydration or polycythemia

17. BLOOD COMPOSITION

18. MATERIALS Blood sample : EDTA or Finger stick blood sample
capillary tube:
heparinized for fingersticks (red tip)
or plain for anticoagulanted blood (blue tip)
Microhematocrit centrifuge
microhematocrit reader
Clay-type tube sealant
Gauze
Alcohol swab
Lancets for capillary puncture

19. (MICRO)HEMATOCRIT TUBE
Glass or plastic
Plain (Blue-ringed) or heparin (Red-ringed) coated
Characteristics:
75 mm long, 1 mm diameter
Sealant

20. MICROHEMATOCRIT CENTRIFUGE

21. Procedure
Fill blood sample up to 3/4 the length of the tube (use 2 tubes for each sample, plain blue-ringed tube for anticoagulated blood, heparinized red-ringed tube for finger stick).

22. Procedure
Seal one end with sealant of clay at a 90°.

23. Procedure
Place the filled and sealed capillary tubes into the centrifuge. The sealed ends should point toward the outside of the centrifuge.
Centrifuge at (11000 rpm) for 5 min
Read Hct in % by using microhematocrit reader
NR :
F %
M 41 – 51%
new born 53%- 65%

24. SEQUENCES OF HCT DETERMINATION
Spin
Place

26. RBC INDICES

27. RBC INDICES RBC indices include: Mean Corpuscular Volume (MCV)
Mean Corpuscular Hemoglobin (MCH)
Mean Corpuscular Hemoglobin Concentration (MCHC)

28. MCV Mean cell volume MCV is average size of RBC MCV = Hct x 10
RBC (millions)
If fL, normal range, RBCs considered normocytic
If < 80 fL are microcytic
If > 100 fL are macrocytic
Not reliable when have marked anisocytosis

29. MCH MCH is average weight of hemoglobin per RBC. MCH = Hgb x 10
RBC (millions)
It’s the weight of Hb in RBCs in the individual blood sample
Normal range:
picogram
>27 : pg Microcytic or normocytic
<32 pg macrocytic

30. MCHC MCHC is average hemoglobin concentration per RBC MCHC = Hgb x 100
Hct (%)
If MCHC is normal, cell described as normochromic
If MCHC is less than normal, cell described as hypochromic
There are no hyperchromic RBCs

31. MCHC 31
Normal values: %
< 38 Not found
> 32 hypochromia