hemoglobin and hematocrit determination
TRANSCRIPT
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Colorimetric Determination of Hemoglobin
Indicator of Fluidity and Transport Capacity
Baldeo, Biendima, Go, Olivar, Soriano
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L-R:PipetteSahli’s StandardHemometer TubeStirring RodDropper
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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)
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Record as
Add drops of water until colors matched
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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
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Conditions Involved
• Anemia• Hypoxia• Leukemia• Cirrhosis• Hypothyroidism• Myeloma
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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
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Improving Sahli’s Method
• Cyanomethemoglobin absorbance reading at 540 nm
• Blood samples treated with:– Potassium ferricyanide– Potassium cyanide– Potassium dihydrogen phosphate
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Total Oxygen-Carrying Capacity (TOC)
• Assumption: 1 g Hb/1.34 mL oxygen
• Male
• Female
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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
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Hematocrit DeterminationIndicator of Blood Viscosity
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Centrifuge blood samples in Eppendorf tubes for 10
min
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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
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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)
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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
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Hematocrit
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Hematocrit and Blood Viscosity
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Conditions
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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)
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Conditions
• Decreased Hematocrit– Hemorrhage– Chronic kidney disease– Inadequate iron uptake– Iron deficiency due to blood loss during menstruation– Anemia– Malnutrition– Overhydration
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Conditions
• A hematocrit of less than 15% can result in cardiac failure
• A hematocrit of over 60% may result in spontaneous blood clotting
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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
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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.
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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.
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Blood IndicesCombining Results for RBC count, Hemoglobin Determination, and
Hematocrit Determination
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Mean Corpuscular Volume (MCV)
• average amount of space occupied by each red blood cell, technically the volume of an RBC
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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
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HIGH MCV
• Liver disease due to alcohol abuse• Myelofibrosis• Reticulocytosis• little vitamin B12 or folic acid• Megaloblastic Anemia
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LOW MCV
• Anemia
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Mean Corpuscular Hemoglobin (MCH)
• reflect the average hemoglobin content of red blood cells– average content (mass, weight) of hemoglobin per
red cell
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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
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Mean Corpuscular Hemoglobin Concentration (MCHC%)
• average concentration of hemoglobin in the RBCs expressed as a percent.
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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
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Indi
cate
d by
MH
C &
MCH
C
Indicated by MCV