Hemoglobin and hematocrit determination


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  • Hb is a chromoprotein, hence the Hb-content in a blood sample may be determined by measurement of its colour.
  • conversion of hemoglobin by hydrochloric acid into acid hematin, which has a brown color in solution
  • The intensity of the color is related to the amount of hemoglobin in the blood sample.The more hemoglobin, the more water required to obtain a color match.
  • Anemia – physiological incapability to replace old RBCs; fatigue, uncontrolled bleeding, higher infection rate; low O2 content in blood to tissues = hypoxiaLeukemia = cancer of lymphatic system and blood-producing tissues (marrow); excessive and abnormal WBCs produced, crowds out RBCs and platelets; low proprotion of hemoglobin.Cirrhosis – liver scarring by hepatitis or alcohol abuse, irreparable; Bile is a yellow-green pigment produced from breakdown of hemoglobin; no bile production = excessive hemoglobin leftover in liver.Hypothyroidism = Insufficient hormone produced by thyroid; eythropoeitin in bone marrow produces RBCs stimulated by thyroid hormones.Myeloma – Kahler’s disease; excessive plasma levels = high antibody production, resulting in low hemoglobin readings in blood <10 g/dL
  • GQ2Acid hematin is not stable and the color gradually starts fading.10 mins minimum
  • GQ 5
  • volume of erythrocytes expressed as a percentage of volume of whole blood in the sample. Also known as packed cell volumeHematocrit separates blood in a tube by centrifugation into: 55% Plasma a straw colored fluid Serum different from plasmaFormed elements [buffy coat, (<1% leucocytes and platelets)] Formed elements [45% erythrocytes]
  • The viscosity of blood increases drastically as the hematocrit increasesThe viscosity of whole blood at normal hematocrit is about 3 -three times as much pressure is required to force whole blood as to force water through the sameblood vessel. When the hematocrit rises to 60 or 70, which it often does in polycythemia, the blood viscosity can become as great as 10 times that of water, and its flow through blood vessels is greatly retarded.
  • Polycythemia – retarded flow of blood in BV; High hematocrit
  • MCV can be falsely elevated in the presence of red blood cell agglutination (as incold agglutinin disease or paraproteinemia) or severe hyperglycemia (glucose > 600mg/dL) as red blood cells become swollenMacrocytic – large RBCS; Microcytic – small; Normocytic - normal
  • Liver Disease – deposition fats / phospolipids in circulating RBCsScarring of the bone marrow means the marrow is not able to make enough blood cells. As a result, the liver and spleen try to make some of these blood cells. This causes these organs to swell ineffective in removing wastes from RBCsOver production of reticulocytes which are 20% larger than normal RBCs (patients with haemolytic anemia)Needed for DNA synthesisImpaired DNA synthesis unaffected RNA synthesis; slow division but rapid deposition of cellular components
  • normal amount of hemoglobin (normal MCHC) are called normochromic.When the MCHC is abnormally low they are called hypochromic, and when the MCHC is abnormally high, hyperchromic.
  • Hemoglobin and hematocrit determination

    1. 1. Colorimetric Determinationof HemoglobinIndicator of Fluidity andTransport CapacityBaldeo, Biendima, Go, Olivar, Soriano
    2. 2. L-R:PipetteSahli’s StandardHemometer TubeStirring RodDropper
    3. 3. Hemoglobin Determination0.1 M HCl at2% mark20 mm3bloodHemolysis ofRBC releasinghemoglobinwhich is thenconverted toacid hematin(Ferrihemechloride)
    4. 4. Add drops of water untilcolors matched
    5. 5. ResultsNormal Levels:Men: 14-17 g/100 mLWomen: 12.5-15 g/100mLInfants: 17-23 g/100 mLVolunteer Hemoglobin Content (g/100mL)Alyssa 13.2Pat 13Cyndi 22Kass 14.2
    6. 6. Conditions Involved• Anemia• Hypoxia• Leukemia• Cirrhosis• Hypothyroidism• Myeloma
    7. 7. Limitations to Sahli’s Method• Standard is not permanent• There is considerable delay in the development ofthe permanent color (many instances reach 20mark)– 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
    8. 8. Improving Sahli’s Method• Cyanomethemoglobin absorbance reading at540 nm• Blood samples treated with:– Potassium ferricyanide– Potassium cyanide– Potassium dihydrogen phosphate
    9. 9. Total Oxygen-Carrying Capacity (TOC)
    10. 10. TOCVolunteer HemoglobinContent (g/100 mL)TOC (mL O2)Alyssa 13.2 563.36Pat 13 473.51Cyndi 22 996.424Kass 14.2 556.57Normal levels:Male: 20 mL O2/ 100 mL bloodFemale: 19 mL O2/ 100 mL blood
    11. 11. Hematocrit DeterminationIndicator of Blood Viscosity
    12. 12. Centrifugeblood samplesin Eppendorftubes for 10min
    13. 13. Hematocrit DeterminationH1H2Where:• H1 = height of the RBCcolumn• H2 = height of the RBC+ height of theplasma column• Calculate Hc%(hematocrit) value
    14. 14. ResultsVolunteer Hematocrit (%)Alyssa 106.67Pat 54Cyndi 57Kass 33Normal 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)
    15. 15. Hematocrit• Normal hematocrit values are:– Newborn: up to 60%– Adults: (males): 40- 54%– (Females): 36 – 46%• Pregnancy: decreased hematocrit, especially inthe last trimester as plasma volume increases• Children: varies with age• Hematocrit = 3x hemoglobin for normal RBCcount and hemoglobin amount only
    16. 16. Hematocrit
    17. 17. Hematocrit and Blood Viscosity
    18. 18. Conditions
    19. 19. Conditions• Elevated hematocrit– Sign of dengue shock syndrome.– Polycythemia vera – bone marrow produces excessivenumbers of RBCs– Chronic obstructive pulmonary diseases– Hypoxia– Capillary leak syndrome– Dehydration (hypovolemia)
    20. 20. Conditions• Decreased Hematocrit– Hemorrhage– Chronic kidney disease– Inadequate iron uptake– Iron deficiency due to blood loss during menstruation– Anemia– Malnutrition– Overhydration
    21. 21. Conditions• A hematocrit of less than 15% can result incardiac failure• A hematocrit of over 60% may result inspontaneous blood clotting
    22. 22. 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
    23. 23. Normal RBC Count = Normal Hematocrit =Normal Hemoglobin Content?• The NORMAL HEMOGLOBIN level does NOTensure NORMAL RBC COUNT andHEMATOCRIT.• NORMAL HEMATOCRIT does NOT ensureNORMAL HEMOGLOBIN content or NORMALRBC COUNT.
    24. 24. Normal RBC Count = Normal Hematocrit =Normal Hemoglobin Content?• Not a definitive test– For example, if youre otherwise healthy and haveno signs or symptoms of illness, results slightlyoutside the normal range on a complete bloodcount may not be a cause for concern, and follow-up may not be needed.
    25. 25. Blood IndicesCombining Results for RBC count,Hemoglobin Determination, andHematocrit Determination
    26. 26. Mean Corpuscular Volume (MCV)
    27. 27. ResultsVolunteer Hematocrit (%) RBC count(millions/mm3)MCV(femtoliters)Alyssa 106.67 6,360,000 176.67Pat 54 8,330,000 64.83Cyndi 57 4,500,000 126.67Kass 33 4,315,000 76.48Normal range: 90-95 µm3 = 90-95 femtoliters
    28. 28. HIGH MCV• Liver disease due to alcohol abuse• Myelofibrosis• Reticulocytosis• little vitamin B12 or folic acid• Megaloblastic Anemia
    29. 29. LOW MCV• Anemia
    30. 30. Mean Corpuscular Hemoglobin (MCH)
    31. 31. ResultsVolunteer Hemoglobin(g/100mLblood)RBC count(millions/mm3)MCH(picograms)Alyssa 13.2 6,360,000 20.75Pat 13 8,330,000 15.61Cyndi 22 4,500,000 48.89Kass 14.2 4,315,000 32.91Normal range: 27-33 picograms
    32. 32. Mean Corpuscular HemoglobinConcentration (MCHC%)
    33. 33. ResultsVolunteer Hemoglobin(g/100mLblood)Hematocrit (%) MCHC(g/100mL)Alyssa 13.2 106.67 12.37Pat 13 54 24.07Cyndi 22 57 38.60Kass 14.2 33 43.03Normal range: 30-34 g/100mL
    34. 34. IndicatedbyMHC&MCHCIndicated by MCV