Complete blood count (CBC):- Part 1 – Differential count (CBC With Platelets), Peripheral blood Smear Interpretation

Complete blood count (CBC)

Sample

1. The best sample is blood in EDTA  (Ethylene diamine tetraacetic acid).
2. Also, prepare fresh peripheral blood smears.
3. This is inexpensive, easy to perform, and rapidly done as a screening test.
4. The sample can be obtained from:
   1. Capillary blood can be obtained from the fingertips, heel, or big toe. This is mostly done in the newborn and infants.
      

      Various sites for the Capillary blood procedure

5. Venous blood can be obtained from the Anticubital vein, veins from the wrist area, or from any area where the vein is prominent.
   

   Venous blood sites on the arm

Indications for CBC:

1. It is a general screening test and gives tremendous information’s about the hematological system and other organ systems.
2. It will differentiate between acute or chronic infection.
3. It will diagnose and type the anemia.
4. It will diagnose any type of leukemia.
5. These are easy, inexpensive, and rapid to perform.
6. It will find any abnormality in the count of platelets.

Precautions:

1. Physical activity and stress may cause an increase in WBCs and the differential values.
2. Pregnancy in the final months may cause an increase in WBC count.
3. Patients with splenectomy have a persistent mild increase in the WBC count.
4. Drugs that may increase the WBC county are:
   1. Aspirin.
   2. Allopurinol.
   3. Steroids.
   4. Quinine.
   5. Epinephrine.
   6. Adrenaline.
   7. Chloroform.
   8. Heparin.
5. Drugs that will decrease the WBC count:
   1. Antibiotics.
   2. Anticonvulsant.
   3. Antimetabolites.
   4. Antithyroid drugs.
   5. Diuretics.
   6. Sulfonamides.
   7. Barbiturates.
   8. Chemotherapy.

CBC (Complete blood count):

1. Complete blood count (CBC) includes:
   1. WBC count is 1 cmm in the peripheral blood.
   2. Differential count on the peripheral blood smears with their concentration.
   3. RBC count and morphology.
2. Peripheral blood smears give a lot of diagnostic information simply stained with Wright and Giemsa stain.
   

   Evaluation by the peripheral blood smear

3. The best area for appreciating the different cells in the peripheral smear is no overlapping of the cells.
4. Hemoglobin gives a reddish-orange appearance to the stained cells.
   

   The peripheral blood smear shows normochromic and normocytic RBCs

4. Definition of complete blood count (CBC):
   1. This is the differential of white blood cells (polys, lymphocytes, monocytes, basophils, and eosinophils).
   2. Morphology of the red blood cells.
   3. Hemoglobin estimation (hemoglobin which carries the oxygen).
   4. Hematocrit.
   5. Platelets count.
5. Peripheral blood smears and studies include:
   1. RBC count.
   2. Hemoglobin.
   3. Hematocrit.
   4. RBC indices.
      1. Mean corpuscular volume (MCV).
      2. Mean corpuscular hemoglobin (MCH).
      3. Mean corpuscular hemoglobin concentration (MCHC).
      4. Red blood cell distribution width (RDW).
6. The White cell differential count includes.
   1. 1. Neutrophil count.
      2. Lymphocytes.
      3. Monocytes.
      4. Eosinophils.
      5. Basophils.
   2. Platelets count.
7. Advantage of CBC:
   1. These are inexpensive, easy, and rapidly can be done as a screening test.
   2. These are the basic workup of any patient with H/O fever or any other problem to advise the complete blood count.

The peripheral blood smear shows:

Red blood cells:

1. 1. The number of red blood cells and their morphology.
   2. There is minimal variation in size, shape, and staining of the RBCs called normocytic cells.
   3. A variation in the size of RBCs is called anisocytosis.
   4. A variation in the shape of RBCs is called poikilocytosis.
   5. Hypochromia is a decrease in the intensity of hemoglobin staining. It is seen when the central pallor of RBCs is >1/3 of the diameter of RBCs.
      

      Peripheral blood smear showing hypochromasia

      Red blood cells abnormalities seen on peripheral blood smear:

      Type of abnormality of RBCs	Characters of RBCs	Importance of abnormality of RBCs
      Microcytosis (microcytes)	RBC diameter  = <6 MCV = <80 fl MHCH = <27%	Thalassemia Iron-deficiency anemia
      Macrocytosis (macrocytes)	RBC diameter = >8 µm MCV = >100 fl	Megaloblastic anemia Hypothyroidism Liver disease (severe)
      Anisocytosis	The variable size of the RBCs	Reticulocytosis Blood transfusion in anemia
      Poikilocytosis	The variable shape of the RBCs	Sickle cell anemia Leukemias Extramedullary hematopoiesis Marrow stress due to any reason
      Spherocytosis	Loss of biconcave shape RBCs No central pallor in the RBCs	Hereditary spherocytosis Increased RBCs destruction by MN system
      Elliptocytosis	RBCs are oval in shape.	Hereditary abnormality Usually harmless
      Acanthocytosis	Irregular spiculated surface RBCs	Liver diseases Abetalipoproteinemia Abnormal membrane lipid contents
      Echinocytosis	Regular spiculated surface RBCs	High plasma free fatty acid Bile acid abnormalities Drugs like salicylates and barbiturates Reversible membrane lipid abnormality
      Target cell (leptocytosis)	Hypochromic RBCs with the small central zone Hb	Thalassemia Obstructive jaundice
      Stomatocytosis	The elongated central zone of pallor in the RBCs	Liver disease (severe) Hereditary defect in membrane transport of Na+
      Hypochromasia	The increased central pallor of the RBCs	Low hemoglobin
      Polychromasia	Presence of RBCs that are not fully hemoglobinized	Reticulocytosis

White blood cells:

1. 1. The number of white blood cells, various types, and their morphology:
   2. WBCs are distinguished from the RBCs by the presence of the nucleus.
   3. The automated machine counts all the nucleated cells RBCs as WBCs, so it needs to be corrected if nucleated RBCs are present in the blood.
   4. Differential white cell count gives the proportion of the different ype of white cells like neutrophils, lymphocytes, eosinophils, monocytes, and basophils.
   5. Visual examination of the stained slides gives the exact picture.
      The differential of white blood cells:

      Type of cells	% in peripheral smear	Absolute values
      Segmented neutrophils	50 to 70	2400 to 75000/cmm
      Band form	2 to 6	100 to 650 /cmm
      Lymphocytes	20 to 40	1000 to 4750 /cmm
      Eosinophils	0 to 4	0 to 450/cmm
      Monocytes	2 to 9	100 to 700 /cmm
      Basophils	o to 2	0 to 200 /cmm

Granulocytes:

1. 1. 1. These are white blood cells,  containing visible granules in the cytoplasm, are the following types:
         1. Neutrophils have neutral staining.
         2. Eosinophils show reddish eosinophilic granules.
         3. Basophils show bluish granules.
         4. Monocytes show a convoluted nucleus and large in size.
            

            Peripheral blood smear showing white blood cells

Neutrophils or polymorphonuclear leukocytes (PMN or polys):

1. 1. 50 to 70% are mature granulocytes (segmented neutrophils) on the peripheral blood smear.
   2. The neutrophils’ cytoplasmic granules react with both basic and acidic stains, producing neutral or light purple granules.
      1. There is a characteristic dense nucleus with 2 to 5 lobes and usually 3 lobes connected by the filament.
   3. The nuclear chromatin is heavily clumped, coarse, or pyknotic, and stains purplish red.
   4. In the mature neutrophils, the Nucleochromatin is condensed into discrete lumps or lobes.
      1. Roughly 6% of neutrophils have one lobe called band form.
      2. 35% have two lobes.
      3. 41% have three lobes.
      4. 17% have four lobes.
      5. 2% have five lobes.
      6. The cytoplasm is pale with irregular outlines containing many fine -pink blue (azurophilic) or grey-blue granules.
         

         Neutrophils showing lobes

      7. The life span of PMN in the peripheral blood is 6 to 10 hours.
      8. Band forms have less mature nuclei.
      9. Neutrophils are actively mobile, and many cells can gather at the site of inflammation or infection called chemotaxis.
   5. Functions:
      1. Neutrophils are the first line of defense against tissue injury or foreign microbes.
      2. Segmentation of the nucleus enables these motile cells to pass through the opening of the endothelium of capillaries and target foreign substances like microorganisms.
      3. Neutrophils may activate the complement system.
      4. Induce immunoglobulin production.
      5. They can do phagocytosis and degrade some particles.
      6. They can produce an enzyme, which can destroy foreign substances ingested or phagocytosed.
      7. The neutrophils cytoplasm contain granules:
         1. Primary granules are myeloperoxidase, acid phosphatase, and other acid hydrolases.
         2. Secondary granules are alkaline phosphatase, lysozyme, and lactoferrin.

Eosinophils:

1. 1. Normal adult blood show 0 to 4% (1 to 5%) in the peripheral blood smear.
   2. Eosinophils are easily recognized by eosinophilic granules in the cytoplasm and affinity the acid eosin stain.
   3. In Wright’s stain, the granules are orange to reddish-orange.
   4. The granules are of uniform size, spherical, and evenly distributed.
   5. Size is slightly larger than the neutrophils and two lobes with condensed chromatin. Rarely can see three lobes.
   6. There is diurnal variation in the eosinophils count, with an increase at night and a decrease in the morning.
   7. The function of eosinophils:
      1. These have a special role in allergic reactions.
      2. These are very important in parasitic defense.
      3. These play a role in the removal of fibrin formed during inflammation.
         

         Eosinophil with prominent granules

Monocytes:

1. 1. In the DLC’s thin areas, these measure 15 to 18 µm and are larger than the neutrophils.
   2. There is abundant cytoplasm in comparison to the nucleus ( N: C = 2:1).
   3. With Wright’s stain, the cytoplasm turns dull grey-blue compared to the pink cytoplasm of the neutrophils.
   4. The nuclei of monocytes may be kidney-shaped, folded, or indented, or occasionally lobulated.
   5. The nucleus shows convolutions like the brain or like a turban.
   6. The shape of monocytes is variable, maybe round, or with blunt pseudopods.
   7. Four important features of the monocytes are:
      1. Nuclear convolutions.
      2. Lacy or Often delicate chromatin.
      3. Dull grey-blue cytoplasm.
      4. Blunt pseudopods.
   8. Digestive vacuoles may be seen in the cytoplasm.
   9. The half-life is 8 hours to 3 days before these cells enter the tissues and become macrophages.
   10. Monocytes are 2 to 9% of the normal blood leucocytes.
   11. Functions:
       1. Defensive mechanism: In different conditions like In infection, bacteria, fungi, pigments, and phagocytosed RBC may be seen in the cytoplasm.
       2. Phagocytosis.
          

          Monocytes in the peripheral smear

Basophils:

1. 1. These cells are rarely found in the peripheral smear, and these are 0 to 2%.
   2. Basophils have large abundant violet-blue or purple-black granules in the cytoplasm.
   3. These granules mostly obscure the nucleus.
   4. These granules measure 0.2 to 1.0 µm. These vary in number, size, and shape—these less numerous than the eosinophil’s granules.
   5. These granules have an affinity for the blue or basic thiazine dyes. Basophil granules are water-soluble.
   6. Basophils show diurnal variation like eosinophils, increasing at night and decreasing in the morning.
   7. Functions:
      1. In the tissue, they become mast cells.
      2. These cells have a receptor for the IgE.
      3. On degranulation, produce histamine.
         

         Basophil with prominent granules

Lymphocytes:

1. 1. Lymphocytes are the second most numerous cells in the peripheral blood smears.
   2. Lymphocytes are 20 to 40% of the adult blood cells.
   3. Lymphocytes are small, varying in size of 7 to 10 µm. There are intermediate and large lymphocytes.
   4. Wright’s stain gives blue cytoplasm varying in intensity from light to dark in different cells.
   5. Most lymphocytes don’t have granules.
   6. The nucleus size in small lymphocytes is just the size of RBCs in the same microscopic field.
   7. The nucleus /cytoplasm ratio is 4:1, and nuclei are round or slightly indented.
      1. Chromatin structure is lumpy or clumped and stains dark purple with the lighter bluish-purple area between chromatin aggregates.
         

         lymphocytes structure

   8. Functions of lymphocytes:
      1. Their main function is humoral immunity.
      2. B-L produces antibody dependant immune reaction.
      3. T-lymphocytes help B-lymphocytes and give rise to cell-mediated immunity.

Platelets:

1. 1. Platelets are produced in the bone marrow by the fragmentation of the cytoplasm of megakaryocytes.
   2. Each megakaryocyte gives rise to 1000 to 5000 platelets.
   3. The size of the platelets is 2 to 4 µm in diameter and varies in shape.
   4. The average number of platelets is 7 to 15/oil immersion field.
      1. Estimate the number of platelets in 10 oil immersion fields.
      2. Platelets are mostly seen in groups.
      3. It is needed to count the number of platelets in the group.
      4. Platelets have no nucleus.
   5. The normal platelets are 250 x 10⁹/L with a range of 150 to 400 x 10⁹/L.
   6. The normal life span of the platelets is 7 to 10 days. About 1/3 of the marrow output of platelets is trapped in the spleen, and it will increase in case of splenomegaly.
      

      Platelets formation

   7. Structure:
      1. The platelets are the smallest structure in the peripheral blood smear, and this measure 3.0 x 0.5 µm in diameter with a mean volume of 7 to 10 fl.
   8. Functions:
      1. The surface coat’s glycoproteins are important in the platelet’s reactions of adhesion and aggregation, which are the initial events in the platelet plug formation in the process of hemostasis.
      2. The plasma membrane phospholipids (known as platelets factor 3) are important and in the conversion of coagulation factor X to Xa and prothrombin (factor II) to thrombin (factor IIa).
      3. The platelets contain three types of granules:
         1. α-granules:
            1. Contains a heparin antagonist.
            2. Platelets-derived growth factor (PDGF).
            3. β-thromboglobulin.
            4. Fibrinogen.
            5. VWF and other factors.
         2. Dense granules contain:
            1. Adenosine diphosphate (ADP).
            2. Adenosine triphosphate (ATP).
            3. 5-hydroxytryptamine (5-HT).
            4. Calcium.
         3. Lysosomes contain:
            1. Hydrolytic enzyme.
            2. Peroxisomes contain catalase.
               

               Platelets function

Summary of the Cytochemistry of various cells found in the peripheral blood:

Types of the cells	Peroxidase	Nonspecific esterase	Periodic Acid-Schiff (PAS)	Acid phosphatase
Segmented neutrophils	Positive (++)	Positive (+)	Positive (++)	Diffusely positive
Band neutrophils	Positive (++)	Positive (+)	Positive (+ to ++)	Diffusely positive
Eosinophils	Positive (++)	Negative	Negative  (Cytoplasm +)	Diffusely positive
Basophils	Partly positive	Negative	Positive (++)	Negative
Monocytes	Negative to positive (- to +)	Positive (++)	Diffusely positive (+)	Positive (++)
Lymphocytes	Negative	Focal locally positive (+) (Acid esterase )	Granules partly  positive (+)	Often positive (+), Fine granules, and focal
Platelets	Negative	Positive (+)	Positive (+)	Positive (+)

Peripheral blood normal values (CBC):

Normal Peripheral blood (CBC):

1. TLC is total leucocytes count:
   1. 4,100 to 10900/cmm.
2. DLC is differential count. Normal values:
   1. Neutrophils = 48 % to 77 %
   2. Lymphocytes = 10 % to 40 %
   3. eosinophils  =  0.3 % to 7 %
   4. Monocytes = 0.6 % to 9.6 %
   5. Basophils = 0.3 to 1 %
3. Platelets = 140,000 to 400,000 /cmm

Hemoglobin:
Normal

Source 2

• Male 14 to 18 g /100 ml
• Female 12 to 16 g/ 100 ml
• Pregnant female = >11 g/dL
• Old people’s values are slightly low.

Source 1

Age	Hb g/dL	Hb g/dL  Male	Hb g/dL Female
Fetal blood
18 to 20 weeks	11.47 ± 0.78
21 to 22 weeks	12.28 ± 0.89
23 to 25 weeks	12.40  ±  0.77
26 to 30 weeks	13.35 ± 1.17
Infants
Cord blood	13.5  to 20.5
1 month	10.7 to 17.1
2 month	9.0 to 13.0
4 month	10.3 to 14.1
9 month	11.4 to  14.0
one year	11.3 to 14.1
2 to 5 year	11.0 to 14.0
5 to 9 year	11.5 to 14.5
9 to 12 year	12.0 to 15.0	Male	Female
12 to 14 year		12.0 to 16.0	11.5 to 15.0
15 to 17 year		11.7 to 16.6	11.7 to 15.3
18 to 44 year		13.2 to 17.2	11.7 to 15.5
45 to 64 year		13.1 to 17.2	11.7 to 16.0
65 to 74 year		12.6 to 17.4	11.7 to 16.1

• For concersion into SI units x 10 = g/L
• Critical value = <5 g/dL  or >20 g/dL

ESR  (Erythrocytes sedimentation rate):
Normal

• Male Older than 50 years = 0 to 15 mm/ hour
• Female = 0 to 25 mm /hour
• Child = upto 10 mm/hour
• Newborn = 0 to 2 mm/hour

Platelet count:

Normal

• Adult = 140,000 to 400,000/cmm
• Children = 150,000 to 450, 000/cmm
• Platelets are assessed by the smear or by a Haematology analyzer.

Neutrophils increased in:

1. Infections.
2. Myocardial infarction.
3. Stress.
4. Metabolic diseases.
5. Inflammations.

Neutrophil Decreased in:

1. In radiation therapy or chemotherapy.
2. Infections.
3. Hypersplenism.
4. Folic acid or B12 deficiency.
5. Hepatic diseases.
6. Drugs
7. collagen vascular diseases.

Eosinophils increased in:

1. Allergy.
2. Parasitic infestation.
3. Skin disorders.
4. Neoplastic diseases like Hodgkin’s lymphoma.
5. Collagen vascular diseases.

Eosinophil Decreased in:

1. Cushing’s syndrome.
2. Stress.

Lymphocytes increased in:

1. Chronic infections.
2. Lymphocytic leukemia.
3. In immune diseases, e.g, Ulcerative colitis.

Lymphocyte Decreased in:

1. Chronic debilitating illness.
2. Immune-deficiency.

Monocytes increased in:

1. Infections.
2. Collagen vascular diseases.
3. Carcinoma.
4. Monocytic leukemia.
5. Lymphoma.

Basophils Increased in:

1. Chronic myelocytic leukemia.
2. Polycythemia vera.
3. Hodgkin’s disease.
4. In some anemias.

Basophil Decreased in:

1. Hyperthyroidism.
2. Stress.
3. Ovulation.

Note: please see more information in peripheral and RBC smear.

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