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The key takeaways are the principle, steps and clinical significance of performing a manual white blood cell count.

The principle is that venous blood is diluted in a solution containing an acid and dye that lyses red blood cells and stains the nuclei of white blood cells, allowing them to be counted under a microscope.

The steps involve collecting blood in a tube, diluting it, charging a hemocytometer slide, allowing cells to settle, and counting cells under a microscope.

General

​G eneral Hematology 
Hematology  
Laboratory Report 
Report  
Based On: White Blood Cell 
Cell 
Count (Manual) 
(Manual) 
   
 

Name of Student : Romanda Greene  


USI # 1019820 
Date: 08 -10-2018
Title: Determination of Total White Blood Cell Count (Manual Method)

Principle:
A manual Leukocyte count represents the number of white blood cells in 1mm3/L of undiluted
whole blood [1,2] and is performed after diluting well-mixed anticoagulated venous blood in a
diluent that contains a weak acid and dye [3] at a specific volume[3] in a test-tube to lyse the
non-nucleated, mature erythrocytes[3] and stains the nuclei of white blood cells.[3] The Lysing
agent reduces the pH of the solution resulting in hemolyzed erythrocytes[4] and converts the
hemoglobin released from the cell into acid hematin, [4] which gives the resulting solution a brown
color.[4] The intensity of the brown color is directly related to the amount of hemoglobin present
in the red blood cells.[4]

❏ Low pH-induced hemolysis of RBCs may be due to entry of the acid in the
cytoplasm of the RBCs and causing oxidative stress on RBC membrane. [5]
❏ At low pH Spectrins of cytoplasm of RBC are precipitated, Resulting in damage.[5]
❏ Due to the reduced pH, the Hemoglobin structure changes. [5]

The Dye or Staining Agent [3,4,5] used -usually Methylene blue or Gentian Violet- is a basic dye
[5] indicates a strong attraction to the nuclei of leukocytes contain deoxyribonucleic acid. [5]
Deoxyribonucleic acid- a double helix- is made up of a negatively charged (phosphate)
backbone.[5] In water, methylene blue has a positive charge,[5] When DNA (​the presence of
negatively charged molecules in the nuclei)[5]​ comes in contact with methylene blue, their
opposite charges attract[5], causing methylene blue's "rings" to slide in between the "rungs" of the
DNA "ladder.”[5] The result: a rich blue-black stain that identifies the location of the cell
nucleus.[5]

The, blood is diluted to a known proportion


-usually- a 1:20 dilution.[3-7] A hemocytometer
is charged with the dilution and placed under a
bright-field microscope to count the number of
cells.[5] The hemocytometer or counting
chamber is composed of two raised surfaces
each in the shape of a 3-mm x 3-mm square [5]
separated by a moat[5] and is subdivided into
16-squares.[5] A coverslip is placed on the top
of the counting surface.[5]The distance between
each counting surface and the cover slip is
0.1mm and total volume is 9mm3. [5]
The count is multiplied by dilution factor and reported as number of cells per microliter (µL) of
whole blood.[5- 7]

Material and Reagent: as per lab handout

Method: as per lab handout

Clinical Significance
Leukocytes fight infection and defend the body by phagocytosis, [1] in which leukocytes encapsulates
[1] foreign organisms and destroy them.[1] In addition, leukocytes also produce, transport, and
distribute antibodies as part of the immune response [1] to a foreign antigen. [1] The leukocytes (white
blood cells) serves as a useful guide to the severity of the disease process. [1]

A WBC count is normally ordered as part of the ​complete blood count (CBC)​, which may be performed
when an individual undergoes a routine health examination.[8] The test may be done when someone
has general ​signs​ and ​symptoms​ of an ​infection​ and/or ​inflammation such​ as: fever, chills, body aches,
pain, headaches. [8]

A WBC count indicates an overall increase or decrease in the number of white blood cells.[8] A
health practitioner will consider the results of a WBC count together with results from other
components of the ​complete blood count (CBC)​ [8] as well as a number of other factors, such as
physical examination, medical history, and ​signs​ and ​symptoms​.[8]

Leukocytosis [1-7] - irrespective of the type of cell (granulocyte, monocytes, lymphocytes) [7]-
is ​ defined as a white blood cell count greater than 11,000 per mm3 (11 ×109 per L),​[1] i​s
frequently found in the course of routine laboratory testing.[1] An elevated white blood cell
count typically reflects the normal response of bone marrow to an infectious or inflammatory
process[1]​- ​systemic or local infection present- as a normal protective response resulting from
liberation of various cytokines[1] that stimulate the development of leukocyte precursors and
release of mature cells from the marrow, endothelium, and spleen [7]and the immune system is
working to destroy an infection.[7]
Pneumonia [9]- causative agents include ​Streptococcus pneumoniae​[10]​, Pneumocystis jiroveci
[10]​- a​ n infection that inflames the air sacs in one or both lungs.[9] The air sacs may fill with
fluid or pus (purulent material)[9], causing cough with phlegm or pus, fever, chills, and difficulty
breathing. [9-10]
Meningitis [9]- causative agents include ​Cryptococcal meningitis​[10]​,Neisseria meningitidis​[10]​,
Herpesvirus[10]​ - ​ an inflammation of the membranes (meninges) surrounding the brain and
spinal cord. [9-10]
Inflammation​ or inflammatory conditions such as R​heumatoid arthritis​[8]- an autoimmune
disease in which the body’s immune system [9]– which normally protects its health by attacking
foreign substances like bacteria and viruses[9] – mistakenly attacks the joints.[9] This creates
inflammation[9] that causes the tissue that lines the inside of joints (the synovium) to thicken,
resulting in swelling and pain in and around the joints. [9]

Occasionally, leukocytosis is the sign of a primary bone marrow abnormality[7-9] in white


blood cell production, maturation or death (apoptosis) related to a leukemia or myeloproliferative
disorder. [7-9]
Chronic Myelogenous Leukemia[12] - Chronic leukemia results from the proliferation and
persistence (lack of apoptosis) of relatively mature blood cells . [12] The bone marrow cells
produce an abnormal protein (the BCR-ABL1 tyrosine kinase),[12] which stimulates chronic
myelogenous leukemia cells to grow and survive better than normal blood cells.[12]

Acute leukemias[12]- the marrow is typically overpopulated with blast cells.[12]They can't carry
out their normal functions, and they multiply rapidly, so the disease worsens quickly. [12]

Hyperleukocytosis-leukostasis syndrome ( > 30,000 cells/mm3) associated with acute myeloid


leukemia (AML). [1,11,12] In these conditions, immature, malignant leukemic cells
pathologically adhere to the microvascular endothelium in the brain and lungs [11] leading to
microvascular occlusion and ischemic stress resulting in cerebral infarction and respiratory
compromise.[11] The leukemic cells release cytokines and toxins, further leading to local tissue
damage.[11]

Leukocytosis can also occur due to the redistribution within the blood white blood cells from the
“marginal”[11] pool are mobilized and poured into the circulating pool. [11] This occur in cases
such as pregnancy, mental stress, physical exercise , infancy, and food intake and digestion and
has no clinical significance.[11]

Leukopenia[1-7]- irrespective of the type of cell (granulocyte, monocytes, lymphocytes)[7] - is


defined as an absolute decrease in the total leukocyte count less than 4,000 cells per mm3
(<4.0x10/L )[1,9] - that may result from decreased marrow production of leukocytic
precursors[9], by peripheral destruction or sequestration of circulating leukocytes[9], or by
autoimmune cellular damage or destruction leading of leukocytes.[9]
Autoimmune Disorders[9] - such as Systemic lupus erythematosus (SLE)[9]- the body fails to
recognise its own cells and begins to attack them.[9] In cases of leukopenia the body’s WBC’s
are perceived as foreign and is attacked leaving the body vulnerable to infections.[10] The
treatments for lupus[10], including corticosteroids[10] and cytotoxic drugs,[10] also destroy
white blood cells, increasing the risk of infections further.[10]

Chemotherapy[13]- In addition to eliminating cancer cells, chemotherapy drugs eliminate normal


cells that divide rapidly[1,7, 13], such as those in the bone marrow that form the different types
of blood cells. [7]Suppression can result in a reduction in white blood cells, resulting in an
increased risk of infections.[7]

Enlarged Spleen (Splenomegaly)[7]- increases the spleen’s mechanical filtering and may start to
remove healthy blood cells as well as abnormal blood cells, reducing the number of healthy cells
in your blood- this includes the destruction of leukocytes- Compensatory bone marrow
hyperplasia occurs in the blood cell lines that are reduced in the circulation. [7]

HIV and AIDS[10]- Human immunodeficiency virus, HIV, causes a life-threatening illness
known as acquired immunodeficiency syndrome, or AIDS.[10] HIV uses CD4 cells, a specific
type of white blood cell known as T helper lymphocytes [10], to replicate itself, producing more
virus particles. [10] This damages and destroys CD4 cells, lowering the white blood cell count
and leaving the immune system vulnerable to infections. [10]The illness AIDS occurs when the
level of CD4 cells falls too low, meaning the body cannot effectively fight infections. [10]

Bruton Agammaglobulinemia[5, 10]-In the absence of BTK, B lymphocytes do not differentiate


or mature.[5,10]The Bruton tyrosine kinase (BTK).mutations underlying X-linked
agammaglobulinemia (XLA) interferes with the development and the function of B lymphocytes
and their progeny.[5] The major block occurs in the development of pro–B cells to pre–B cells
and then to mature lymphocytes.[5] Patients can have pre–B cells in the marrow, but they have
few, if any, functional (mature) B cells in the peripheral blood and the lymphoid tissues.[5]

Under Physiologic condition[9] , a decrease in total leukocyte count is unusual and rare. [8,9]
Exposure to extreme cold , even under arctic condition and in spite of acclimatization may
reduce the count to only slightly below (< 4,000 /mm3) level. [9]

Leukopenia due to any cause increases the risk for developing a pyogenic and other infection,
since,the body is not capable of fighting against infections.[9] Prolonged leukopenia of more
than 15 days increases the risk of many other life-threatening infections. [9]

Testing may be performed when there are signs and symptoms that a health practitioner thinks
may be related to a blood disorder, ​autoimmune disorder​, or an immune deficiency.[8]
A WBC may be ordered often and on a regular basis to monitor an individual who has been
diagnosed with an infection, blood or immune disorder or another condition affecting WBCs.[8]
It may also been ordered periodically to monitor the effectiveness of treatment or when a
particular therapy is known to affect WBCs, such as radiation or chemotherapy.[8]
When WBC counts are used for monitoring purposes, a series of WBC counts that continues to
rise or fall to abnormal levels indicates that the condition or disease is getting worse.[8] WBC
counts that return to normal indicate improvement and/or successful treatment.[8]
Limitations
In certain diseases (eg. measles ,pertussis and sepsis) , the increase of leukocytes is so great
that the blood picture suggests leukemia. [1] leukocytosis of a temporary nature (leukemoid
reaction) must be distinguished from leukemia. In leukemia, the leukocytes is permanent
and progressive. [1]

Occasionally,leukocytosis / Leukopenia is found when there is no evidence of clinical


disease.[1,9] Such finding suggest the presence of :

❏ Eating (A heavy meal prior to test) and physical exercise may cause an increase in
White blood cell count.[9]

❏ Any stressful situation that leads to an increase in endogenous epinephrine production


and a rapid rise in leukocyte count.[1]
❏ Pregnancy( final month) and labour may be associated with an increase in white
blood cell levels. [9]
❏ Patients who had an splenectomy have a persistent mild to moderate elevation of
white blood cell count.[9]
❏ The white blood cell count tend to be lower in the morning and high in the late
afternoon (an early morning low and late afternoon high peak).[1]
❏ The white blood cell count tend to be age related :
➔ Normal newborns and infants tend to have higher white blood cell count than adults .
[10]
➔ It is not uncommon for the elder to fail to respond to infection by the absence of
Leukocytosis. The elderly may not develop an increase in white blood cell even in
the face of severe bacterial infection. [5]
❏ Drugs that cause increased white blood cell include adrenaline, allopurinol,
aspirin,triamterene , chloroform, epinephrine and steroids. [10]

❏ When corticotropin (adrenocorticotropic hormone) is given to a healthy person,


leukocytosis occurs. [1]
❏ When ACTH is given to a patient with severe infection, the infection can spread rapidly
without producing the expected leukocytosis; therefore what would normally be an
important sign is obscured. [1]
❏ Any nucleated erythrocytes present in the sample are not lysed by the diluting fluid
resulting in false elevated WBC count. [1]
❏ Exposure to Sunlight in​creases the number of white blood cells in the blood.[1]
❏ Drugs that cause a decrease in white blood cell level include antibiotics,
anticonvulsants, antihistamine, antimetabolites, antithyroid drugs, arsenicals,
chemotherapeutic drugs, diuretics and sulfonamide. [5]

Sources of Error:
Erroneous results can be caused based on the following:

Technical Error
❏ Improper collection of blood specimens. [1]
❏ Improper volume measurement of blood and diluent.[3-7]
➔ If the sample is not dissolved well, the cells become crowded and will be difficult
to count.[5]
➔ If it is too dissolved, the sample size will not be enough to develop strong
inferences about the concentration in the original mixture.[5]
❏ Improper charging of the chamber. Uneven flow of diluted blood into the chamber results
in irregular distribution of cells. [3-7]
➔ Overfilling the chamber of the hemacytometer, which causes erroneously high
counts.[5]
➔ Underfilling the chamber of the hemocytometer causes low count. [5]
❏ Use of defective pipette ,wet or dirty pipette.[3-7]
❏ Not allowing cells to settle for an adequate amount of time. [3]
❏ Poor pipetting technique causes high or low counts.[3-7] Poor pipetting technique
includes:
➔ Undershooting pipette with blood.[7]
➔ Overfilling pipette with blood. [7]
➔ Air bubbles in the shaft. [5]
➔ Not mixing the blood specimen thoroughly. [5-7]
❏ Not mixing the diluted specimen prior to filling the Hemacytometer.[3-7]
❏ Improper counting [3-7]
❏ Wrong calculation [1]
❏ Non-uniform suspensions due to improper mixing. [6]
❏ Dirty or scratched Hemocytometer. [3-7]
Errors inherent in the method
❏ Error of visual count [3-7]
➔ One potential error is mistaking dirt or clumped red blood cell debris( artifacts)for
leukocytes. [3]
❏ Uneven distribution of cells in the counting chamber causes erroneous results.[6]

Observation/ Results:

Field Number WBC/MM3

1 94 Cells

2 95 Cells

3 94 Cells

4 96 Cells

Total Cells in field 376 Cells

Area of 4 WBC square = 4 x 0.1=4mm2


Volume of 4 WBC squares = 4x 1/10 =0.4mm3
Dilution Factor 1:20
Volume correction factor = volume desired
​volume used

= 1.0​ = 2.5
0.4

Therefore, cells in 1 mm3 volume of diluted blood = 376 x 2.5


Therefore, cells in 1mm3 volume of undiluted blood = 376 x2.5 x 20 = 18,800 cells/ mm3
The white blood cell count (18,800 cells/ mm3) is above normal range ,thus, indicates
Leukocytosis
.
Normal Range:

Black Adults 3.2-10.0x103 /cells/mm3 or 3.2-10.0x109/L or 3200-10,000 cells/mm3 [1]


Adults 4.5-10.5x103/cells/mm3 or 3.2-10.0x109/L or 4200-10,000 cells/mm3 [1]

Children

0-2 weeks 9.0-30.0x103/cells/mm2 or 9.0-30.0x109/L or 9000-30,000 cells/mm3 [1]


2-8 weeks 5.0-21.0 x10 3/cells/mm3 or 5.0-21.0x109/L or 5000-21,000 cells/mm3 [1]
2 months- 6 years 5.0-19.0x10 3/cells/mm3 or 5.0-19.0x109/L or 5000-19,000 cells/mm3 [1]
6 years- 18 years 4.8-10.8 x 10 3/cells/mm3 or 4.8-10.8 [1]

Critical Values < 500 cells/mm3 or > 30,000 cells/mm3 [2]

Quality Control
Collection of Sample
❏ A disposable syringe and needle (or vacutainer) should be used to draw the
blood.[112,13]
❏ The puncture site should be clean with alcohol.[ 1,12,13]
❏ Disposable gloves should always be use to prevent contamination. [1,12,13]
❏ The vein should be made prominent before making a puncture.[13]
❏ The needle should be held at an angle of 30o -40o and introduced into the vein steadily
and firmly. [1,12,13]
❏ The tourniquet should should be removed and before taking the needle out of the vein to
prevent hematoma formation. [12]
❏ Ensure that there is no leakage from the specimen (if collected in bottle or bag). [13]

Pipetting
❏ Pipette should be clean , dry and without chip or broken tips.[11,13]
❏ The tip of pipette should drop into the blood drop, otherwise while sucking blood into
pipette air bubbles enter along with blood. [11,13]
❏ The tip of pipette should be wiped off otherwise , the extra blood enters the pipette along
with the diluting fluid is sucked. [11,13]
❏ Blood in test tube should be diluted immediately, otherwise it will clot. [11,13]
❏ Contaminated diluting fluid should not be used. Blood should not be allowed to get into
diluent because this will affect subsequent cell count with the same diluent.[11,13] Tip of
the pipette should be dip inside the diluting fluid throughout the process of filling,
otherwise air bubble enter the pipette.[13]

Hemocytometer
❏ The content in the test-tube must be thoroughly mixed before charging. [13]
❏ The pipette, coverslip and Neubauer Chamber should be dry and thoroughly cleaned.
[1,11,13]
❏ Do not overcharge or undercharge the chamber: if overfilled or underfilled , the chamber
must be cleaned and recharge. [1,11,13]
❏ After chamber is filled allow the cells to settle for 2 minutes. A pause longer than 1-2
minutes allow fluid to start evaporating and causes inaccuracies in the count. [13]
Conclusion:

Reference:
1. Frances TF, Marshall BD. A manual of Laboratory Diagnostic Test. 7th Edition. Lippincott
Williams and Wilkins.2004 pg.132-133
2. Daniels R. Delmar’s Guide to Laboratory and Diagnostic Tests.2nd Edition. New York,
New York. Delmar Cengage Learning.2010 pg. 394-395
3. Yash PA; Samir LA;David NA; Finberg KE;et.al.Laboratory Medicine: The Diagnosis of
Disease in the Clinical Laboratory. 2nd Edition. Weitz M ;Yoo C. China. McGraw-Hill
Education.2014 pg 415-420
4. Turgeon ML. Linné & Ringsrud’s Clinical Laboratory Science: THE BASIC AND
ROUTINE TECHNIQUES .6ed. Elsevier (Mosby) 2011.pg
5. Anderson D. Lab Values: Everything You Need to Know about Laboratory Medicine and
its Importance in the Diagnosis of Diseases EBook.1st. edition. Creations.2018 pg.

6. Pagana KD. Mosby's Manual of Diagnostic and Laboratory Tests.Pagana TJ. Elsevier
Health Sciences. 2013

7. McPherson RA​; ​Pincus​ MR. Henry's Clinical Diagnosis and Management by Laboratory
Methods EBook. 23rd Edition. Elsevier. 2017
8. The American Association for Clinical Chemistry (AACC). White Blood Cell Count.
Schaeffer,K. AACC Publishers.2017.
https://labtestsonline.org/tests/white-blood-cell-count-wbc
9. Pal​ GK. ​Textbook of Practical Physiology. ​Illustrated Edition. Orient Blackswan. 2001.
pg
10. Rodak​ BF; ​George​ AF;​ Doig k. Hematology​: Clinical Principles and Applications.
Illustrated Edition. Elsevier Health Sciences. 2007. pg.

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