Colorimetric Determination of Hemoglobin

Indicator of Fluidity and Transport Capacity

Baldeo, Biendima, Go, Olivar, Soriano

L-R:PipetteSahli’s StandardHemometer TubeStirring RodDropper

Hemoglobin Determination

0.1 M HCl at 2% mark

20 mm3 blood

Hemolysis of RBC releasing hemoglobin which is then converted to acid hematin (Ferriheme chloride)

Record as

Add drops of water until colors matched

Results

Normal Levels:Men: 14-17 g/100 mL Women: 12.5-15 g/100mLInfants: 17-23 g/100 mL

Volunteer Hemoglobin Content (g/100 mL)

Alyssa 13.2Pat 13

Cyndi 22Kass 14.2

Conditions Involved

• Anemia• Hypoxia• Leukemia• Cirrhosis• Hypothyroidism• Myeloma

Limitations to Sahli’s Method

• Standard is not permanent• There is considerable delay in the development

of the permanent color (many instances reach 20 mark)– 10 minutes: 95% acid hematin– 20 minutes: 98% acid hematin– 1 hour: 100% acid hematin

• Large errors have been found in Sahli’s pipette, recalibration is needed before using them

Improving Sahli’s Method

• Cyanomethemoglobin absorbance reading at 540 nm

• Blood samples treated with:– Potassium ferricyanide– Potassium cyanide– Potassium dihydrogen phosphate

Total Oxygen-Carrying Capacity (TOC)

• Assumption: 1 g Hb/1.34 mL oxygen

• Male

• Female

TOCVolunteer Hemoglobin

Content (g/100 mL)TOC (mL O2)

Alyssa 13.2 563.36Pat 13 473.51

Cyndi 22 996.424Kass 14.2 556.57

Normal levels:Male: 20 mL O2/ 100 mL bloodFemale: 19 mL O2/ 100 mL blood

Hematocrit DeterminationIndicator of Blood Viscosity

Centrifuge blood samples in Eppendorf tubes for 10

min

Hematocrit Determination

H1

H2

Where:• H1 = height of the RBC

column• H2 = height of the RBC

+ height of the plasma column

• Calculate Hc% (hematocrit) value

ResultsVolunteer Hematocrit (%)

Alyssa 106.67Pat 54

Cyndi 57Kass 33

Normal Values:Children, 9-11 years (whole blood): 34%-43% (0.34-0.43) Females, 12-14 years (whole blood): 34%-44% (0.34-0.44)Males, 12-14 years (whole blood): 35%-45% (0.35-0.45)Females, 15-17 years (whole blood): 34%-44% (0.34-0.44)Males, 15-17 years (whole blood): 37%-48% (0.37-0.48)

Hematocrit

• Normal hematocrit values are: – Newborn: up to 60%– Adults: (males): 40- 54% – (Females): 36 – 46%

• Pregnancy: decreased hematocrit, especially in the last trimester as plasma volume increases

• Children: varies with age • Hematocrit = 3x hemoglobin for normal RBC

count and hemoglobin amount only

Hematocrit

Hematocrit and Blood Viscosity

Conditions

Conditions

• Elevated hematocrit– Sign of dengue shock syndrome.– Polycythemia vera – bone marrow produces excessive

numbers of RBCs– Chronic obstructive pulmonary diseases– Hypoxia– Capillary leak syndrome– Dehydration (hypovolemia)

Conditions

• Decreased Hematocrit– Hemorrhage– Chronic kidney disease– Inadequate iron uptake– Iron deficiency due to blood loss during menstruation– Anemia– Malnutrition– Overhydration

Conditions

• A hematocrit of less than 15% can result in cardiac failure

• A hematocrit of over 60% may result in spontaneous blood clotting

Technical Errors

• Results increased in : – Strenuous exercise – High altitude

• Technical errors– Inadequately mixed blood sample– Length of time spent and speed of centrifuge– Prolonged venous status during venipuncture

Normal RBC Count = Normal Hematocrit = Normal Hemoglobin Content?

• The NORMAL HEMOGLOBIN level does NOT ensure NORMAL RBC COUNT and HEMATOCRIT.

• NORMAL HEMATOCRIT does NOT ensure NORMAL HEMOGLOBIN content or NORMAL RBC COUNT.

Normal RBC Count = Normal Hematocrit = Normal Hemoglobin Content?

• Not a definitive test– For example, if you're otherwise healthy and have

no signs or symptoms of illness, results slightly outside the normal range on a complete blood count may not be a cause for concern, and follow-up may not be needed.

Blood IndicesCombining Results for RBC count, Hemoglobin Determination, and

Hematocrit Determination

Mean Corpuscular Volume (MCV)

• average amount of space occupied by each red blood cell, technically the volume of an RBC

ResultsVolunteer Hematocrit (%) RBC count

(millions/mm3)MCV

(femtoliters)Alyssa 106.67 6,360,000 176.67

Pat 54 8,330,000 64.83Cyndi 57 4,500,000 126.67Kass 33 4,315,000 76.48

Normal range: 90-95 µm3 = 90-95 femtoliters

HIGH MCV

• Liver disease due to alcohol abuse• Myelofibrosis• Reticulocytosis• little vitamin B12 or folic acid• Megaloblastic Anemia

LOW MCV

• Anemia

Mean Corpuscular Hemoglobin (MCH)

• reflect the average hemoglobin content of red blood cells– average content (mass, weight) of hemoglobin per

red cell

ResultsVolunteer Hemoglobin

(g/100mL blood)

RBC count (millions/mm3)

MCH (picograms)

Alyssa 13.2 6,360,000 20.75Pat 13 8,330,000 15.61

Cyndi 22 4,500,000 48.89Kass 14.2 4,315,000 32.91

Normal range: 27-33 picograms

Mean Corpuscular Hemoglobin Concentration (MCHC%)

• average concentration of hemoglobin in the RBCs expressed as a percent.

ResultsVolunteer Hemoglobin

(g/100mL blood)

Hematocrit (%) MCHC (g/100mL)

Alyssa 13.2 106.67 12.37Pat 13 54 24.07

Cyndi 22 57 38.60Kass 14.2 33 43.03

Normal range: 30-34 g/100mL

Indi

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MH

C &

MCH

C

Indicated by MCV