• Information
  1. Some Normal Blood Values
  2. Normal Red Blood Cells (RBC) Values
  3. Iron and Iron Metabolism
  4. Leukocytes (White Blood Cells, WBC)
  5. Platelets
  6. Coagulation

• TERMINOLOGY AND DEFINITIONS
  1. Coombs' Test
  2. Dohle Body
  3. Howell-Jolly Bodies (HJB)
  4. Haptoglobin
  5. Heme
  6. Hemopexin
  7. Hemosiderin
  8. Heinz Bodies
  9. Polychromasia

• MISCELLANEOUS DISORDERS
  1. Monoclonal Gammopathy
  2. Methemoglobinemia

A. Some Normal Blood Values

1. Blood (arterial) pH 7.35-7.45 corresponds to proton levels [H+] = 45-35 nM
2. Total Body Water: 60% of weight. 42kg in a 70kg person
   1. Intracellular: 40% (2/3 of total), 28 liters (L) in 70kg person
   2. Extracellular: 20% (1/3 of total), 14 L in 70kg person
3. Extracellular Body Water is divided into:
   1. Interstitial 16.5% (~4/5) 11.5 L
   2. Plasma 3.5% (~1/5) 2.5 L
4. Total Blood Volume ~2X Plasma Vol 5.0 L
5. Plasma oncotic pressure
   1. Total 28mm 6-7.5 g/dL
   2. Albumin 21mm 3.5-4.5 g/dL
   3. Globulins 6.5mm 2.5-3 g/dL
   4. Fibrinogen 0.3mm 0.3 g/dL
6. Plasma Osmolality 280-295 mOsm/L

B. Normal Red Blood Cells (RBC) Values

1. Hematocrit (HCT): M 38-54% F 36-47%
2. Hemoglobin (HB): M 14-18g% F 12-16g% Child: 12-14g% Newborn: 14-24g%
3. Erythrocytes (per mm3): M 4.5-6 million F 4.3-5.5 million
4. Reticulocytes (per mm3): 0-1.5%
5. Diameter: Adult: 5.5-8.8 µm Newborn: 8.6 µm
6. Mean Corpuscular Volume (MCV): 80-90 cµm (fL) Newborn: 100-110 cµm
7. Mean Corpuscular Hemoglobin (MCH): 27-32 pg
8. Sedimentation Rate (ESR): M 0-9mm/hr F 0-20mm/hr [age dependent]
9. RDW (red cell distribution width): Normal <14.5%; RDW >14.5% called anisocytosis

C. Iron and Iron Metabolism

1. Normal plasma levels: F 70-180 g/dL, M 55-160 g/dL
2. Normal total body iron ~1gm
3. Iron Losses
   1. No active excretion of iron
   2. Slight loss of iron due to very mild gastrointestinal losses
   3. Women lose iron with blood loss during menses
   4. Therefore, pre-menopausal women require more iron than men
   5. Tissue damage occurs at 28g total body iron
   6. This iron level is equivalent to ~100 units red cells transfused
4. Summary of Metabolism
   1. Iron is mainly contained in hemoglobin (Hb) and myoglobin proteins
   2. Iron is overwise stored in cells bound to a 500K protein called Ferritin
   3. Major iron transporter in blood is called Transferrin
   4. Blood normal Total Iron Binding Capacity (TIBC) 47-70µMol (mainly transferrin)
   5. Ferritin Levels: F 15-300 g/L; M 25-400 g/L
   6. Note that ferritin levels increase in inflammatory processes (acute phase reactant)
5. Erythrocyte Iron Metabolism
   1. Major Iron Metabolism in red blood cells (RBC) through free Fe, Heme, and Hb
   2. Serum protein Haptoglobin transports Hb
   3. Normal haptoglobin level 100-300 (50-200mg/dL)
   4. Serum protein Hemopexin transports Heme moiety
   5. Normal hemopexin level 0.9-3.8 g/L
6. Iron Overload
   1. Hemosideridosis is generalized or focal iron overload
   2. Iron overload with organ damage is called Hemochromatosis
   3. Hemochromatosis requires total body iron > 15gm
   4. As total body iron levels increase, so do levels of ferritin, the iron storage protein
7. Iron Toxicity is marked at ferritin levels >3000 g/L
   1. Mainly endocrine toxin
   2. Adrenal, pancreatic islets and parathyroid glands most affected initially
   3. Hepatitis with Liver failure, Cardiomyopathy (infiltrative) occurs also
8. Increased RBC lysis
   1. Normal RBC lifetime is ~120 days
   2. Decreased lifetime in hemolytic anemia and splenomegaly
   3. Both haptoglobin and hemopexin decrease with increased RBC lysis
9. Active removal of iron
   1. Iron requires chelation for reduction of body stores
   2. Urinary excretion occurs after chelation

D. Leukocytes (White Blood Cells, WBC) [2]

1. Total WBC: 5000 - 10,000/µL (100%)
2. Neutrophils: 3000 - 6500/µL
   1. Segmented PMN: 2500 - 6000µL (40-60%)
   2. Band (immature) neutrophils: 0 - 500/µL (0-5%)
   3. Myelocytes (normal): 0/µL
3. Eosinophils: 50 - 300/µl (1-3%)
4. Basophils: 0 - 100/µl (0-1%)
5. Lymphocytes: 1200 - 4000/µL (20-40%)
6. T Lymphocytes: 1100 - 3600/µL (90%)
   1. CD4+ T cells: 800 - 2000/µL (60%)
   2. CD8+ T cells: 300 - 1200/µL (30%)
   3. Natural Killer (NK) Cells: 50 - 400/µL (10%)
7. B Lymphocytes: 100 - 400/µL (10%)
8. Plasma Cells: 0 - 50/µL
9. Monocytes: 200 - 800/µL (4-8%)

E. Platelets

1. Normal levels are 150,000 - 350,000 / µL
   1. Serious bleeding usually only seen with levels <20,000 / µL
   2. Increased thrombotic events with levels >800,000-1,000,000 / µL
2. Lifetime in blood: t1/2 = 3-4 days; mean lifetime 7-10 days
3. Thrombopoietin and other growth factors responsible for generation

F. Coagulation

1. Clotting time (venous) 6-10 min (Lee and White) 10-30 min (Howell)
2. Prothrombin time (mainly evaluates extrinsic pathway) 9-11 seconds
3. Partial thromboplastin time (mainly evaluates intrinsic pathway): 26-37 seconds

1. This is a test for detecting antibodies (Abs) directed against RBCs
2. Also called antiglobin test
3. Abs prepared in animals directed against specific human serum proteins (ie. IgG, C3)
4. These Abs agglutinate RBC if these human serum proteins are present on the RBC surface
5. Ability of anti-IgG or anti-C3 antisera to agglutinate patient's RBC is Direct Coombs Test
6. Indirect Coombs Test
   1. Incubating Rh and ABO compatible RBC with a patient's serum, then
   2. Performing a Coombs test (ie. add Coombs' reagent) on these RBC
   3. Detects anti-alloantibodies, ie. antibodies against non-self RBC

B. Dohle Body

1. Inclusions, round or oval bodies up to 2µm
2. Found in neutrophils from patients with infections, burns, trauma, pregnancy, or cancer
3. Often accompanied by "toxic granulation."

C. Howell-Jolly Bodies (HJB)

1. Spherical (eccentric) granules, ~1µm diam
2. Seen in stroma of circulating erythrocytes (especially stained preparations)
3. Probably represent nuclear remnants
4. Normally removed by the spleen
   1. HJB are present in patients with splenectomy or auto-infarcted spleens
   2. Common in sickle cell disease because the spleen has infarcted

D. Haptoglobin

1. Plasma protein which binds hemoglobin (see above)
2. Conserves iron released from red cell destruction
3. Tetramer with two alpha and two ß chains.
   1. The ß chain shows 30% homology to serine proteases
   2. Three common variants of human haptoglobin, differing in the alpha chains
4. Reduced in cases of hemolytic anemia
5. However, haptoglobin is also an acute phase inflammatory protein
   1. When inflammation is present, level may be elevated
   2. Therefore, only a low level of haptoglobin is informative for hemolysis evaluation

E. Heme

1. Iron protoporphyrin
2. When iron is not complexed, free protoporphyrin is formed
3. Free protoporphyrin levels increase in Fe deficiency and lead poisoning

F. Hemopexin

1. Serum ß-globin class protein which binds hemin (oxidized heme moiety) in 1:1 fashion
2. Complex is cleared by liver parenchymal cells slowly
3. Decreased in cases of hemolytic anemia
4. Hemopexin is not an acute phase protein and levels are fairly stable during inflammation

G. Hemosiderin

1. Intracellular protein/iron complex, including ferritin
2. Usually found in monocytes and macrophages
3. When renal epithelia are sloughed, hemosiderin may enter urine

H. Heinz Bodies

1. Brilliant cresyl blue staining bodies in RBCs
2. Occur due to RBC instability
3. Seen in some red cell deficiencies and hemoglobinopathies

I. Polychromasia

1. Darker colored red cells (ie. purple), either reticulocytes or new mature RBC's
2. Increased polychromasia on smear usually means increased RBC formation
3. Most commonly in reponse to increased RBC loss

1. Normally, immunoglobulins (Ig) are polyclonal with no major bands on electrophoresis
2. Occasionally, clonally selected B lymphocytes will proliferate out of proportion to others
3. These cells differentiate to plasma cells and produce a single Ig
4. This Ig will appear on serum protein electrophoresis (SPEP) and is monoclonal in origin
5. Finding of a high level band(s) on SPEP is diagnostic of monoclonal gammopathy
6. Multiple myeloma and Waldenstrom's macroglobulinemia are classic neoplastic gammopathies
7. Other hematologic malignancies such as lymphoma, chronic B cell lymphocytic leukemia (CLL) cause monoclonal gammopathies

B. Methemoglobinemia [3]

1. Methemoglobin is formed by oxidation of iron moiety of Hb, from normal Fe2+ (ferrous) to abnormal Fe3+ (ferric)
   1. Ferric (3+) heme cannot bind oxygen and causes allosteric chanages in remaining heme
   2. These allosteric changes impair release of oxygen and shifts oxyHb dissociation curve left
   3. Methemoglobin normally reduced back to ferrous state by NADH-cytochrome b5 reductase
2. Normally, methemoglobin occurs in ~1% of total heme (~0.1-0.15gm/dL)
3. Cyanosis occurs at methemoglobinn levels of 1.5gm/dL (~10% of Hb)
4. Three Main Types
   1. Acquired: due to a variety of drugs and toxins
   2. Hemoglobin M: amino acid substitution at or near heme group
   3. Reduced activity of erythrocytic NADH-cytochrome-b5 reductase (congenital recessive)
5. Methemoglobin associated with reduced oxygen saturation on pulse oximetry
   1. Shows normal level of calculated oxygen saturation on arterial blood gas
   2. Hb electrophoresis normal unless HbM variant present
6. Drugs associated with Methemoglobinemia
   1. Rare consequence of exposure of normal red cells to various oxidizing drugs
   2. Benzocaine
   3. Dapsone
   4. Nitrates
   5. Sulfonamides
7. Treatment
   1. Oxygen
   2. Riboflavin (vitamin B2) and ascorbic acid (vitamin C) for mild disease
   3. Methylene blue for severe disease
   4. Exchange transfuion and hyperbaric oxygen for refractory, life-threatening disease
8. Methylene Blue
   1. Acts as cofactor to increase rate of NADPH dependent methemoglobin reduciton
   2. Given intravenously 1-2mg/kg for severe methemoglobinemia
   3. Usually for methemoglobin >30% of signs/symptoms of methemoglobinemia
   4. Methylene blue is ineffective with low NADPH levels such as glucose-6-phosphate dehydrogenase (G6PD) deficiency
   5. Higher doses of methylene blue act as oxidants and can worsen condition

References

1. Winkelstein A and Kiss JE. 1997. JAMA. 278(22):1982
2. Stock W and Hoffman R. 2000. Lancet. 355(9212):1351
3. Hurford WE and Kratz A. 2004. NEJM. 351(4):380 (Case Record)