BASIC HEMATOLOGIC TESTS

SBO1.Review the functions and compositions of blood

HEMOGLOBIN (H b ) A. Characteristics:

Oxygen carrying compound in RBC Amount of Hb/ 100ml of blood

Normal values:* for Men : 14 – 18 gm/100 ml* for women : 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 : 20 gm/ 100 ml* For Children : 10 – 13 gm/100 ml

B.Changes in Hemoglobin (not due to blood loss or polycythemia)

Increased in:o High WBC Counto Heavy Smoking as a result of

compensationo Dehydration (as a result of

hemoconcentration) Decreased in:

o Childreno Recumbent from upright positiono Pregnancyo Diurnal variation (evening): the Hb count

is higher when taken in the morningo African Americans: may be due to Iron

deficiency anemiao Female sexo Intravenous fluids

RED BLOOD COUNT (RBC COUNT)- Is the number of RBCS / mm3 give an indirect

estimate of HB content

Normal values* For Men : 4.5 – 6.0 x 10 12 / L* For Women : 4.0 – 5.5 x 10 12 / L

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

Decreaed in:o Hemorrhageo Anemia (due to production defect or

increased intravascular hemolysis)o Acute intake of intravenous fluid

HEMATOCRIT (H ct ) 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 : 40 – 54 %* For Women : 37 – 47 %

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)

FormulaMCV = HCT

RBC Count

Causes of Increased MCVo Folate or b12 deficiencyo Chronic liver diseaseo Chronic alcoholismo Cytotoxic chemotherapyo Cardiorespiratory abnormalityo Reticulocytosis o Myelodysplastic syndromeo Unexplained

Subject: Pathology (CP)Topic: Basic Hematologic TestLecturer: Dr. CagampanDate of Lecture: 8.15.2011Transcriptionist/Editor: DressPages: 9

o Normal newborn

Causes of Decreased MCVo Chronic iron deficiencyo Alpha or beta thalassemia o Anemia of chronic disease

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

Normal Values:MCHC = 31 – 37 %

FormulaMCHC = Hb (g/L)

Hematocrit

Conditions that affect MCHC Increased :

o Spherocytosis o High titer cold agglutininso Free plasma hemoglobino Severe plasma lipemia o Heavy smoking

Decreased :o Chronic iron deficiencyo Sideroblastic anemiao 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)

FormulaMCH = Hb (g/L)

RBC Count (10 12 / L)

REMEMBER: Increased in MCH = Macrocytosis

Decrease MCH = Microcytosis & Hypochromia

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

*Anisocytosis – varied sizes of RBC

- RDW elevated in Iron , Folate, B12 deficiency, RBC fragmentation, homozygous hemoglobinopathies

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 WBC’s

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 reticulocytesWright stain will stain a hue of blue pink for POLYCHROMATOPHILIA

NORMAL VALUESReticulocyte : 0.5 – 1.5 %

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

iron deficiency

WBC COUNT- Can be done through manual or automatic

methods

Normal Values

WBC = 5 – 10 x 10 9 / L

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

Note: if nucleated RBC are present, take note of percentage of nucleated RBC per 100 WBC

Normal WBC differential values

Neutrophils 50 – 70 %Band / Segmenter 0 – 5 %Metamyelocytes 0 – 1 %Lymphocytes 20 – 40 %

(increased in children)Monocytes 0 – 7 %Eosinophils 0 – 5 %Basophils 0 – 1 %

**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

BONE MARROW ASPIRATION- Usually done on the iliac crest

o Aspirate o Put on slideo Do an imprint of the bone marrow coreo Stain with the wright staino 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 SMEAR1. Place a drop of blood on a slide2. With another slide, let the blood touch the edge

of the vertical slide3. 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 cells5. Another technique, the other slide is positioned

at a 30 degree angle

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

CELLS IN THE BLOOD SMEAR

A. NORMAL PERIPHERAL BLOOD SMEAR (PBS) With small central pallor

B. ABNORMALITIESa. Anisocytosis : different sizes of RBC

b. Poikilocytosis : difference in shape of RBC’s

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

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

f. Megalocyte

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

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

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

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 RBC’s undergoing hemolysis due to G6PD deficiency (where there is an absence or reduced glutathione).

“mukhang pacman”

l. Schistocyte a. Fragmented red cellsb. Seen usually in microangiopathic

hemolytic anemias (narrowed blood vessels)

“cells become fragmented due to the abnormally narrowed blood vessels as the blood traverses”

due to the defect in the cytoskeleton

m. Crenated RBCa. can be normal; may be an artifactc so

be careful in evaluating this. b. Looks like a Burr cell with Spinec. Common name : Artifactual

n. Elliptocytes a. Seen in congenital elliptocytosisb. < 5 - 10 % of RBC’s can normally

appear like elliptocytesc. “ellipsoid”

o. Reticulocytea. Granules are seen when stained by

Cresyl blue stain

p. Sickle Cell a. caused by a point mutation in the β-

globin chain of haemoglobin, causing the hydrophilic amino acid glutamic acid to be replaced with the hydrophobic amino acid valine at the sixth position

q. Spherocytea. 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.

Treatment : Splenectomy

r. Target cella. Also called “leptocyte”b. Due to the uneven hemoglobinization

and distribution of Hbc. 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

RBC contracts to its most surface-tension efficient and least flexible configuration

Spherocytes

e. Common in hemoglobinopathies and post splenectomy

s. Tear drop cella. Seen in Myeloid Metaplasia or chronic

myeloproliferative disorders (10

myelofibrosis)

t. Stomatocytea. Central pallor looks like a slitb. If in abundance, may be due to a liver

disease but it is not specific

u. Siderocytea. Iron granules in RBC (in bone marrow)b. Stain : Prussian Bluec. For iron stores

v. Nucleated Rbca. Usually counted per 100 WBCb. Can be seen in new bornsc. Appear as a small nuclei with

abundant cytoplasm

w. Rouleaux formationa. RBC’s appear as stack of coinsb. Usually happens when you have

increased protein c. Seen in Hypergammaglobulinemia,

multiple myeloma (due to the light protein called Bence Jones)

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 hemoglobine. 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

y. Howell Jolly Bodiesa. 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 celle. May appear after splenectomyf. Remnants of DNA

z. Papenheimer bodiesa. IRON RELATED, and a special stain is

neededb. 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 hemosiderind. 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

BB. BASOPHILIC STIPPLING : seen in lead poisoninga. Multiple, uniform, evenly distributed

dark dots [arrows]i. Diffuse – appears as fine blue

dustingii. Coarse – much more outlined and

easily distinguished.iii. Punctuate – coalescing of smaller

forms; very prominentb. 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

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“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.SIf you want to have bigger and clearer picture browse on doc cagampans powerpoint

Disclaimer:

This transcription is an aid to help us study; it does not substitute the book, please read your SBO and try to answer it. This transcription is based on recording and powerpoint of the lecturer.