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
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.
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
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
Hematocrit DeterminationIndicator of Blood Viscosity
Centrifugeblood samplesin Eppendorftubes for 10min
Hematocrit DeterminationH1H2Where:• H1 = height of the RBCcolumn• H2 = height of the RBC+ height of theplasma column• Calculate Hc%(hematocrit) value
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)
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
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)
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 incardiac failure• A hematocrit of over 60% may result inspontaneous 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 NOTensure NORMAL RBC COUNT andHEMATOCRIT.• NORMAL HEMATOCRIT does NOT ensureNORMAL HEMOGLOBIN content or NORMALRBC COUNT.
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.
Blood IndicesCombining Results for RBC count,Hemoglobin Determination, andHematocrit Determination