Subject: Pathology (CP) Topic: Basic Hematologic Test Lecturer: Dr. Cagampan Date of Lecture: 8.15.

2011 Transcriptionist/Editor: Dress Pages: 9

BASIC HEMATOLOGIC TESTS SBO 1.Review the functions and compositions of blood HEMOGLOBIN (Hb) A. Characteristics: Oxygen carrying compound in RBC Amount of Hb/ 100ml of blood Normal values: * for Men : * for women : Decreaed in: o Hemorrhage o Anemia (due to production defect or increased intravascular hemolysis) o Acute intake of intravenous fluid

14 18 gm/100 ml 12 16 gm / 100ml

boys and girls have same normal values range till 11 y/o In newborns, capillary Hb, Hct higher than venous blood Normal values * For Newborn : * For Children :

HEMATOCRIT (Hct) Percentage of RBC mass to original blood volume After centrifugation, in a MICROHEMATOCRIT TEST TUBE, height of RBC column is measured and compared with height of original column of original whole blood Automated counters measured from RBC count and MCV (mean corpuscular volume) Normal Values: * For Men : * For Women :

40 54 % 37 47 %

20 gm/ 100 ml 10 13 gm/100 ml

B.Changes in Hemoglobin (not due to blood loss or polycythemia) Increased in: o High WBC Count o Heavy Smoking as a result of compensation o Dehydration (as a result of hemoconcentration) Decreased in: o Children o Recumbent from upright position o Pregnancy o Diurnal variation (evening): the Hb count is higher when taken in the morning o African Americans: may be due to Iron deficiency anemia o Female sex o Intravenous fluids RED BLOOD COUNT (RBC COUNT) - Is the number of RBCS / mm3 give an indirect estimate of HB content Normal values * For Men : * For Women :

Useful relationships between Hb,Hct, RBC count : Hb x 3 = Hct RBC ( millions ) x 3 = Hb RBC ( millions ) x 9 = Hct

RBC INDICES - Indices that incorporate in the RBC count the hemoglobin concentration and the hematocrit A. Mean Corpuscular Volume (MCV) - MCV = HCT/RBC count - Uses the effect of the average RBC size on Hct Normal Values * For Both Male & Female : 80 100 fL (fL = femtoliters = 10 -15 L) Formula MCV

HCT RBC Count

4.5 6.0 x 10 12 / L 4.0 5.5 x 10 12 / L

Increased in: o Polycythemia (Polycythemia Vera) o Relative Polycythemia

Causes of Increased MCV o Folate or b12 deficiency o Chronic liver disease o Chronic alcoholism o Cytotoxic chemotherapy o Cardiorespiratory abnormality o Reticulocytosis o Myelodysplastic syndrome o Unexplained o Normal newborn

Causes of Decreased MCV o Chronic iron deficiency o Alpha or beta thalassemia o Anemia of chronic disease

*Anisocytosis varied sizes of RBC RDW elevated in Iron , Folate, B12 deficiency, RBC fragmentation, homozygous hemoglobinopathies

REMEMBER: Increase MCV = increase RBC Decrease MCV = Microcytic Anemia B. Mean Corpuscular Hemoglobin Concentration (MCHC) - Estimates the average concentration of Hb in the average RBC - Depends on the relationship of the amount of Hb to volume of RBC MCHC= Hb/Hct

REMEMBER: * RDW normal in Thalassemia minor, anemia of chronic disease, etc. * RDW never decreases * Increase in RDW = more varied the size of the RBC PERIPHERAL BLOOD SMEAR Allows visual estimation of the amount of Hb in RBC, size and shape of RBC See abnormal / immature cells Good estimate of platelet count (7-25 plates /OIF) NOTE: Best sample is from Capillary blood (through finger puncture) - because the cells are not distorted, and there are no anticoagulants that will alter the RBCs and the WBCs RETICULOCYTE COUNT Increased in hemolytic anemia RETICULOCYTE - Stage after the normoblast, Anucleated and contains nuclear remnants and is LARGER THAN RBC - nucleus is extruded with cytoplasmic microsomes or ribosomes which remain for 1-2 days - Large than rbc with dark blue dots or thin short irregular linear structures NOTE: Methylene or cresyl blue stain is used to demonstrate reticulocytes Wright stain will stain a hue of blue pink for POLYCHROMATOPHILIA NORMAL VALUES Reticulocyte

Normal Values: MCHC = 31 37 % Formula MCHC

Hb (g/L) Hematocrit

Conditions that affect MCHC Increased : o Spherocytosis o High titer cold agglutinins o Free plasma hemoglobin o Severe plasma lipemia o Heavy smoking Decreased : o Chronic iron deficiency o Sideroblastic anemia o Anemia of chronic disease C. Mean Corpuscular Hemoglobin (MCH) - Based on estimates of the quality of Hb in the average RBC - MCH = Hb/RBC count - Influenced by size of RBC - Depends on the amount of Hb in relation to size of cell Normal Values: MCH = 26 34 pg (pg = pictogram = 10 -12 g) Formula MCH

0.5 1.5 %

Hb (g/L) RBC Count (10 12 / L)

Most common causes of reticulocytosis - Hemolytic anemia, chronic or acute - Acute bleeding - After treatment of vitamin b12, iron deficiency

Folate,

REMEMBER: Increased in MCH = Macrocytosis Decrease MCH = Microcytosis & Hypochromia

WBC COUNT - Can be done through manual or automatic methods Normal Values WBC = 5 10 x 10 9 / L

D. Red Blood Cell Distribution Width (RDW) - Cell counting machines can calculate an index of the RBC size difference (*anisocytosis) using data from the histogram and MCV

Nucleated RBC counted as WBC * usually present in Newborns and Anemic patients

NEUBAUER COUNTING CHAMBER - Like a slide with thick grid on top - L = squares where you count your WBCs

Note: if nucleated RBC are present, take note of percentage of nucleated RBC per 100 WBC Normal WBC differential values Neutrophils Band / Segmenter Metamyelocytes Lymphocytes Monocytes Eosinophils Basophils 50 70 % 05% 01% 20 40 % (increased in children) 07% 05% 01% CELLS IN THE BLOOD SMEAR A. NORMAL PERIPHERAL BLOOD SMEAR (PBS) With small central pallor

**take note of the bold ones ** PLATELET COUNT Manual or machine counting Normal Values: Platelet :

150 400 x 10 9 / L

PBS PLATELET ESTIMATE : 7 25 platelets / OIF *OIF = Oil Immersion Field 3 8 platelets / 100 RBC

Formula: Platelet count = # of platelet in 10 OIF x 2000 B. ABNORMALITIES a. Anisocytosis : different sizes of RBC

BONE MARROW ASPIRATION - Usually done on the iliac crest o Aspirate o Put on slide o Do an imprint of the bone marrow core o Stain with the wright stain o Processed to see activity of bone marrow To confirm diagnosis of megaloblastic anemia, leukemia, Multiple Myeloma Diagnosis of bone marrow hypoplasia To document deficiency in body iron stores To demonstrate metastatic / infectious disease

HOW TO MAKE A SMEAR 1. Place a drop of blood on a slide 2. With another slide, let the blood touch the edge of the vertical slide 3. Move back a little and make a sweeping movement towards the other end (from one end to other movement) 4. It should be gentle so as not to destroy the cells 5. Another technique, the other slide is positioned at a 30 degree angle

b. Poikilocytosis : difference in shape of RBCs

e. Macrocyte : bigger than lymphocytes, and sometimes may also be hyperchromic

f.

Megalocyte

c. Hypochromia Has a bigger central pallor Graded depending on the size of pallor (ex bigger central pallor will be graded as MODERATE or SEVERE)

d. Microcyte To determine the size of the RBC, use Lymphocyte as a reference

g. Anisochromia : there is a difference in color (pale, dark and etc.) Ex. Of Patient with ANEMIA Some may be reticulocytes = reticulocytosis

h. Polychromasia: stained with methylene blue and if you see remnants of ribosomes these are reticulocytes

deficiency (where there is an absence or reduced glutathione). mukhang pacman

i.

Acanthocytes : thorny cells, which are found in non-specific diseases Ex. Liver and Renal diseases

l.

Schistocyte a. Fragmented red cells b. Seen usually in microangiopathic hemolytic anemias (narrowed blood vessels) cells become fragmented due to the abnormally narrowed blood vessels as the blood traverses

j.

Burr cells : spines are more even and equally distributed in the entire circumference Burr cells = seen in Renal Disease Ex. Microangiopathic anemias like DIC, SLE, malignant hypertension, thrombocytic purpura m. Crenated RBC a. can be normal; may be an artifactc so be careful in evaluating this. b. Looks like a Burr cell with Spine c. Common name : Artifactual

k. Bite Cells a. are seen in G6PD Deficiency Bite Cells = seen in G6PD Deficiency = Heinz Bodies *Heinz Bodies result of Hemoglobin (Hb) precipitation and are recognized by the spleen. The Hemoglobin (Hb) precipitation is due to RBCs undergoing hemolysis due to G6PD

n. Elliptocytes a. Seen in congenital elliptocytosis b. < 5 - 10 % of RBCs can normally appear like elliptocytes c. ellipsoid

q. Spherocyte a. Lack of central pallor b. auto-hemolytic anemia characterized by the production of red blood cells (RBCs) that are sphere-shaped, rather than bi-concave disk shaped c. defect in 1 or more of the proteins of the RBC cytoskeleton, including, spectrin and ankyrin. RBC contracts to its most surfacetension efficient and least flexible configuration

due to the defect in the cytoskeleton

Spherocytes

o. Reticulocyte a. Granules are seen when stained by Cresyl blue stain

p. Sickle Cell a. caused by a point mutation in the globin chain of haemoglobin, causing the hydrophilic amino acid glutamic acid to be replaced with the hydrophobic amino acid valine at the sixth position

Treatment r.

Splenectomy

Target cell a. Also called leptocyte b. Due to the uneven hemoglobinization and distribution of Hb c. Ex. In liver disease, there is a phospholipid acyl transferase activity abnormality so it resulted tto an imblanace between surface ratio and membrane itself d. L- cat problem e. Common in hemoglobinopathies and post splenectomy

s. Tear drop cell a. Seen in Myeloid Metaplasia or chronic myeloproliferative disorders (10 myelofibrosis)

v. Nucleated Rbc a. Usually counted per 100 WBC b. Can be seen in new borns c. Appear as a small nuclei with abundant cytoplasm

t.

Stomatocyte a. Central pallor looks like a slit b. If in abundance, may be due to a liver disease but it is not specific

w. Rouleaux formation a. RBCs appear as stack of coins b. Usually happens when you have increased protein c. Seen in Hypergammaglobulinemia, multiple myeloma (due to the light protein called Bence Jones) u. Siderocyte a. Iron granules in RBC (in bone marrow) b. Stain : Prussian Blue c. For iron stores

x. Heinz Bodies a. Due to G6PD deficiency

Cont.. Heinz bodies; b. Large (0.3 to 2 micrometer) rigid inclusions that severely distort the membrane c. Stain pale pink or purple with crystal violet and brilliant cresyl blue vital stains. d. Denatured hemoglobin e. May be seen in Alpha thalassemia, G6PD deficiency under oxidant stress, and in any of the unstable hemoglobin syndromes. f. May be seen in red cell injury due to chemical insult

z. Papenheimer bodies a. IRON RELATED, and a special stain is needed b. Clusters of small blue coccoid bodies (arrow) located at the periphery of the cell. i. They are smaller than Howell Jolly bodies. c. They contain hemosiderin d. Found in sideroblastic anemia any condition leading to hemochromatosis or hemosiderosis, hemoglobinopathies and in splenectomized patients

aa. Hb H bodies: seen in Hb H disease

y. Howell Jolly Bodies a. Small 1-2mm,, red purple, eccentrically located, coccoid nuclear fragments (arrow) b. Seen in severe types of hemolytic anemias and megaloblastic anemia, splenectomy or functional hyposplenia and thalassemia c. Seen during absence or reduction of splenic function, or with accelerated or abnormal erythropoiesis, since the pitting action of splenic macrophages cannot keep pace with inclusion formation. d. Single dots present one per cell e. May appear after splenectomy f. Remnants of DNA

bb. BASOPHILIC STIPPLING : seen in lead poisoning a. Multiple, uniform, evenly distributed dark dots [arrows] i. Diffuse appears as fine blue dusting ii. Coarse much more outlined and easily distinguished. iii. Punctuate coalescing of smaller forms; very prominent b. Consist ribonucleoprotein and mitochondrial remnants. c. May be found in any condition showing defective or accelerated heme synthesis, lead intoxication, and thalassemia.

cc. Post splenectomy slide

-------------END OF TRANSCRIPTION ---------Greetings! Hi kay Bee, K, quen (the birthday girl), el.oh, Jez (M.I.A lagi), anna OSO and Dress (congrats for making it to the TOP TEN of Pharma.. wuhoo!!!) ur 5th row family in SEC A is proud of you may you serve as an inspiration to all of us. Ay, oo nga pla.. hi din kay GEM GEM and papabear!! : ) We know that all things work together for the good of those who love God; thos who are called according to HIS purpose. Romans 8:28 P.S If you want to have bigger and clearer picture browse on doc cagampans powerpoint
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