Lecture 4 The Complete Blood Count (CBC) o The Complete Blood Count (CBC) should include: A total red

red blood cell count and hematocrit A total white blood cell count Blood smear examination Differential WBC count, RBC morphology, platelet estimation, and reticulocyte count when patient is anemic Plasma protein concentration

o When proper instrumentation is available, the following values should be gathered for a CBC as well: Hemoglobin concentration MCH (mean corpuscular hemoglobin) MCHC (mean corpuscular hemoglobin concentration) MCV (mean corpuscular volume)

Differential vs. Total vs. Absolute WBC Count o After calculating the Total WBC count with the hemacytometer, and the Differential Percentage Count on your blood smear, the Absolute Differential Counts can be determined. o The Absolute Differential Counts are the Total WBC count times the Differential Percentage counts. o EXAMPLE:

Total WBC count = 10,350 WBC/ul Differential Percentage counts = 79% Neutrophils, 13% lymphocytes, 5% monocytes, 3% Eosinophils, 0% basophils

Absolute WBC Counts Absolute neutrophil count = 10,350 x 79% = 8,177 neutrophils/ul Absolute lymphocyte count = 10,350 x 13% = 1,346 lymphocytes/ul Absolute monocyte count = 10,350 x 5% = 518 monocyte/ul Absolute eosinophil count = 10,350 x 3% = 311 eosinophils/ul Absolute basophil count = 10,350 x 0% = 0 basophils/ul

Hemoglobin Determination Spencer Hemoglobinometer o Examine the blood chamber to the right: it consists of two pieces of glass and a metal clip. Note that one of the pieces of glass has an H-shaped depression cut on it, while the other piece is flat on both sides o A drop of blood is placed on one side of the side containing the H shaped depression o The blood is stirred with the end of the hemolysis applicator until the blood appears as a transparent red rather than a cloudy liquid. o The flat piece of glass is positioned on top of the blood plate and both are slid into the metal clip. The blood chamber is then slid into the slot on the side of the hemoglobinometer and a reading is taken o Hemoglobin Determination: Procedure When the blood chamber has been filled with hemolyzed blood, it is

inserted into the slot on one side of the hemoglobinometer as shown in the accompanying image (insert, upper right). When the hemoglobinometer is held to the eye, and the light switch button is depressed, a green split field appears (insert, upper left). To practice taking a reading, click below and watch as the slide moves along, taking note of the concentration (in grams of hb/100 mL) when the tow halves of the field appear to be the same shade of green. Plasma Protein Concentration o Plasma Contains Albumin Globulin Fibrinogen

o Serum Contains Albumin Globulin

o A low plasma or serum protein value = hypoproteinemia o A high plasma or serum protein value = hyperprotienemia Fibrinogen Determination o Clotting method One blood sample is collected in an anticoagulant (sample 1) and a separate sample is collected without anticoagulant (sample 2) Plasma protein readings are made on the plasma of sample 1 and the

serum of sample 2 The reading on sample 2 is subtracted from the reading on sample 1 for the fibrinogen value. o Precipitation method Two separate blood samples are centrifuged Plasma protein determined in sample 1; sample 2 is placed in a heated water bath for 3 minutes then recentrifuge. Plasma protein determination made on sample 2 and is subtracted from sample 1 for fibrinogen value. Anemia o Anemia is a broad term used to describe an overall decrease in the oxygen carrying capacity of blood this may be due to: An abnormally low amount of blood in circulation Low hemoglobin in RBCs (nutrition) A decreased number of red blood cells (RBCs)

o Anemia itself is not a disease but a sign or symptom of an underlying disease or condition. It is important to find the cause of the anemia as well as to determine the type of anemia. o Anemia can be diagnosed a number of ways: A low hematocrit (<37%) A low total RBC count (<5.5 million/ul) A low hemoglobin concentraion

Total hemoglobin (<12 g/dl) Mean corpuscular hemoglobin concentration (<32 g/dl)

External clinical signs of anemia

o External Clinical Signs of Anemia Fatigue, loss of energy, weakness Shortness of breath, tachycardia Pale mucous membranes

o Classifying Anemia Bone marrow response Red blood cell size Hemoglobin concentration of the RBCs

o Classification of Anemia Bone Marrow Response

o Regenerative Anemia An anemia with an adequate increased presence of immature RBCs (reticulocytes) is a regenerative anemia. A regenerative anemia means the bone marrow is properly doing its job of increasing the production of new RBCs in light of increased demand, which includes the production and release of larger number of reticulocytes Blood smears usually feature prominent polychromasia (reticulocytes), possible nucleated RBCs, and an increased MCV (mean corpuscular volume) Two major causes of a regenerative type anemia are hemorrhage and hemolysis o Reticulocytes The hallmark of a regenerative anemia is an increased in reticulocytes in the blood. This is true for all species EXCEPT the horse; horses donot release reticulocytes into the bloodstream under normal or anemic circumstances Normal Staining polychromatophilia New Methylene Blue reticulocytes

o Reticulocyte Count

The degree of regeneration can be judged by doing a reticulocyte count. Mix few drops blood with equal drops of methylene blue stain in a tube and mix and stand for 10 minutes, then make blood smear on slide and count reticulocytes.

Reticulocyte count procedure: Using a smear stained with new methylene blue or brilliant cresyl blue, the # reticulocytes seen per 1000 RBCs are counted Correction of reticulocyte count for anemia o Reticulocyte % x PCV / normal mean PCV o EX. 15% x 30% / 45% = 10% Absolute reticulocyte count o RBC count X reticulocyte % o EX. 6,000,000 RBC count x 5% = 300,000 ret/ul

o Blood Loss Anemia Blood loss anemia results from the loss or removal of blood from the vascular system Blood loss can be internal or external, and can be due to hemorrhage or hemolysis Blood loss anemias are generally regenerative, given there has been sufficient time (at least 3 days) for the bone marrows response to be observed. Blood loss anemia is usually accompanied by a reduction in total protein. The reduced total protein is due to loss of blood proteins along with cells during hemorrhage as well as dilution of proteins in the blood vessels by fluids drawn into the blood in an effort to maintain blood pressure. Most Common Causes of Blood Loss Anemia Traumatic hemorrhage Parasites (fleas & hookworms) Clotting disorders (genetic and toxins) Immune-mediated conditions

o Hemolytic Anemia Hemolytic Anemia results from any disorder in which the red blood cells are destroyed prematurely (hemolysis) Hemolytic anemia is generally the result of either: An attack on the RBCs by their own antibodies (immune mediated

hemolysis), or Direct damage of the RBC membrane by drugs, toxins, or parasites (Anaplasma, Cytauxzoon, Babesia). There are two types of hemolysis: Intravascular hemolysis, in which RBC rupture in the bloodstream, releasing their hemoglobin into the plasma or Extravascular hemolysis, where RBCs are removed by phagocytosis in the spleen and liver without the release of their hemoglobin into the plasma. Intravascular Hemolysis Intravascular hemolysis occurs when damage to the RBC membranes lead to RBC lysis (breakage) in the bloodstream and release of free hemoglobin into the plasma The released hemoglobin often leads to hemoglobinemia (red plasma or serum) and hemoglobinuria (red urine). Patients may be icteric as well. Icterus is seen in both extra and intravascular hemolysis. Drugs, toxins, and parasites which cause direct cell damage generally result in the intravascular type of hemolysis Direct Cell Damage Some drugs and toxins can structurally change the RBC cell membranes making them more susceptible to hemolysis. Heinz body formation in cats given acetaminophen causes cell fragility

and increases the likelihood of rupture. (Heinz Body=bite in cell) Parasitic anemia (such as Anaplasmosis in cattle) can damage the RBC membranes leading to their intravascular rupture. In Disseminated Intravascular Coagulation (DIC), fibrin strands from small clots can snag and damage RBCs, leading to their hemolysis. (DIC = Death is Coming!!!!) o Immune Mediated Hemolytic Anemia (IMHA) Immune mediated hemolytic anemia (Autoimmune Hemolytic Anemia) is caused by the destruction of RBCs by the animals own immune system. Antibodies are directed against antigens (normal or foreign) on the surface of the RBCs, resulting in the cells removal or rapture. When antibodies are directed against normal components of the RBC the anemia is primary autoimmune hemolytic anemia. When antibodies are directed against a non-RBC antigen stuck on the cell surface (a virus, part of a druc or part of a neoplastic cell), the hemolytic anemia is called secondary autoimmune hemolytic anemia. The most common form of red cell destruction in immune-mediated hemolytic anemia is the extravascular removal of RBC that have antibodies on their surface. The antibodie-coated RBCs are removed by the mononuclear phagocytic system in the spleen, bone marrow and liver. When extravascular removal of RBCs predominates, hemoglobinemia and hemoglobinuria are generally not seen. Intravascular hemolysis is less common in immune-mediated hemolytic

anemia, but it does occur occasionally. Dogs are the most common domestic species affected. Female dogs are affected more often than males, and Cocker spaniels appear to be affected more than other breeds of dogs. Spherocytes (spherocytosis) are one of the hallmarks of autoimmune hemolytic anemia. Some animals with immune-mediated hemolytic anemia will have intravascular agglutination of RBC. This agglutination may be seen grossly in some patients as visile clumping of RCs in a drop of blood as soon as the sample is drawn. Agglutination occurs due to immunoglobin on the cell surface. o Isoimmune Hemolytic Anemia Isoimmune disease is the destruction of RBC of newborns due to differences in blood types between dam and offspring. In isoimmune hemolytic anemia, the antibodies attacking the RBCs are from the mother, and are not made by the animals own immune system. Seen primarily in horses, less commonly in dogs and cats. This type of hemolytic anemia may occur in kittens with type A blood born to queens with type B blood, sired by males with type A blood. o Equine Neonatal Isoerythrolysis Equine neonatal isoerythrolysis (NI) is a form of isoimmune disease in which foals are born healthy, but develop a possibly life-threatening hemolytic anemia within hours to a few days after the ingestion of their

mares colostrum. This condition occurs as a result of hypersensitivity reaction between the mares antibodies in the colostrum and inherited antigens from the sire that are present on the foals red blood cells. o Non-Regenerative Anemia Non regenerative (degenerative) anemia denotes an inadequate bone marrow production for an increased demand for red blood cells Can be associated with nutritional deficiencies (Iron, B vitamins) Bone marrow suppression o Drugs and certain infectious agents such as FeLV o Myelophthistic disorders: The components of marrow are being crowded out by abnormal cells. Most often due to neoplasia of one or more of the cells that normally exist in the marrow. (cancer in bone marrow) o Anemia of chronic renal disease Decreased erythropoietin production

The reticulocyte count, despite a marked anemia, is 60,000ul

Classifying Anemia ***MIDTERM!!!!! Red blood cell Size (MCV) o Normocytic Red cells are of normal size Secondary to chronic disorders (cytic=size)

o Macrocytic

Red cells are larger than normal Seen early in regenerative anemia due to the presence of many reticulocytes

o Microcytic Red cells are smaller than normal Primarily seen in iron deficiency

Hemoglobin Concentration (MCHC) (chromic= color) o Normochromic Red cells have normal color (normal amount of Hb)

o Hypochromic Red cells have a lighter color (less than normal amount of Hb) (hypochromasia) Seen when there are prominent reticulocytes and in iron deficiency o Hyperchromic Theoretical does not happen

Combining Size and HB Concentration o Normocytic, normochromic anemia Normal size RBCs with normal RBC Hb concentration Chronic conditions CRF, GI bleed, FELV

o Macrocytic, hypochromic anemia

Enlarged RBCs with decreased RBC Hb concentration Regenerative anemia due to reticulocytosis, AIHA

o Microcytic, hypochromic anemia Small RBCs with decreased RBC Hb concentration Erythrocytic Indices o Erythrocytic indices are calculated values which gives some information on the average size of RBCs and the average amount of hemoglobin in each RBC. These values are: Mean Corpuscular Volume (MCV) To calculate o MCV = hematocrit(%) x 10 RBC count (millions/mm3 blood) Mean Corpuscular Hemoglobin (MCH) Mean Corpuscular Hemoglobin Concentration (MCHC) o o Examples To calculate MCHC = hemoglobin (g/100mL) x 100 Hematocrit (%) Iron deficiency anemia, lead toxicity

Hematocrit = 38% RBC count = 4.5 million (4.5 x 106) Hemoglobin = 4 g/dL

o MCV = 38 x 10 = 8.44 fL (femtoliters) 4.5 o MCHC = 4 x 100 = 10.52 g/dL 38 o MCV Normal MCV = normocytic High MCV = macrocytic Low MCV = microcytic

o MCHC (and MCH) Normal MCHC = normochromic High MCHC = hyperchromic (does not happen) Low MCHC = hypochromic

o Reference Values MCV (fL) Dog 60-77 (70) Cat 39-55 (45) Horse 34-58 (46) Cow 40-60 (52)

MCHC (g/dL) Dog 32-36 (34)

Cat 30-36 (33) Horse 31-37 (35) Cow 30-36 (32)

Hb (g/dL) Dog 12-28 (15) Cat 8-15 (12) Horse 11-19 (15) Cow 8-15 (12)

o Interpreting CBC Data A 12 week-old puppy is presenting to the veterinary clinic because of lethargy and bloody diarrhea. The puppys condition has been going down hill for the past 2 weeks. Initial examination reveals a rough hair coat, dehydration, increased heart and respiratory rates. Blood is taken for a CBC and a feacal exam is done. The results are as follows: Blood smear: o 65% Neutrophils; 12% lymphocytes; 8% Monocytes; 15% Eosinophils; 0% Basophils o Platelets were adequate numbers. o The RBCs all appeared to be of normal size, shape and color, with no inclusions or parasites noted. PCV: 25% Reticulocytes were <1%

RBC Hemocytometer count = 550 = ?? RBCs/uL WBC Hemocytometer count = 180 = ?? WBCs/uL Hemoglobinometer reading = 4 g/dL Total plasma protein = 5.5 g/dL Fecal exam revealed the presence of numerous hookworm ova The total RBC count = 5.5 x 106 /uL anemia PCV of 25% anemia The hemoglobin reading of 4 g/dL anemia The total WBC count = 19,800/uL leukocytosis Absolute WBC differential: o 12,870 neutrophils mild neutrophilia o 2,376 lymphocytes normal o 1,584 moncytes mild monocytosis o 2,970 eosinophils marked eosinophilia

MCV = 25(PCV) x 10 = 45.44 fL microcytic anemia 5.5(RBC count) MCHC = 4(hgb) x 100 = 16 g/dL hypochromic 25(PCV) Reticulocytosis of <1% which such marked anemia indicates the bone marrow IS NOT responding non-regenerative anemia

The total protein value of 5.5 g/dL is low hypoproteinemia

Conclusions and comments: The anemia is non-regenerative, microcytic, hypochromic.

The presence of hookworm ova and blood diarrhea indicates a blood loss anemia.

With chronic blood loss, iron is lost so the bone marrow does not have enough raw materials to make more RBCs with enough hemoglobin to respond to the anemia.

With blood loss anemia, plasma proteins are lost along with the RBCs, so the total protein is low (hypoproteinemia)

There is a mild leukocytosis, caused mostly be the marked eosinophilia. Eosinophilia is a common reaction to parasitic infections. The mild neutrophilia and monocytosis are common, nonspecific reactions to most disease states.

Laboratory #4 o Exam Review

Midterm is from lecture 1to 4 Remember the calculations that you need to memorize!!! Know safety Need to know microscope parts and its functions Know the simple things about microscope like how to clean it and what the oil is used for Know the three different microscopes Know our instruments Know all blood collection contains and what is its use Know the different white blood cells and the purpose of the cell Know the components of blood

Know the wrights stain, the order and name and the time (30-60-30) Know red blood cells Know the morphology of the cells Know terminology Know the artifacts