Manual counting of WBC.

I have the chance to count the WBC  manually  using microscope for practicing purpose.

I counted on different types of WBC in the blood smear and add up until 100.

( I did not follow the name on top but follow the reference book order which provided by my mentor)

* 70 Neutrophil , 0 Basophils,  0 Eosinophils, 24 Lymphocytes, 6 Monocytes *

And then, I compare with the result calculated by the analyzer.

In conclusion, both of us got the similar result.

Reticulocyte Count

Procedure:

1. 3 drops of blood + 1 drop of Brillant Cresyl Blue , and incubate for 15 minutes.
2. After incubation, use the solution to make one thin blood film and view under microscope.

( count 1000 RBC )


[ Eg: If 1 field has roughly 100 RBC means have to look at 10 different fields. ]

Blood Smear

Blood smear/ Blood film.

How to do a Blood Smear?

1. Place a small drop of blood at one end of the smear to be made on the slide.
2. Position the spreader (we use cover slip here) at 45° angle and backing into the drop of blood.

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Differentiation of gender based on Blood Smear.

Female and male  have same amount of neutrophils bearing appendages. ( A to D form )

However, female has more A-form (drumstick) appendages while male has more C-form (tag) appendages due to higher androgen level in men.

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References:

1. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3781649/

Leishman stain

Thin blood smear.

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Leishman stain

• Acid-base staining
• Acid (Red)    - bind to cytoplasm
• Base (Blue)   - bind to nucleic acid (RNA, DNA...)

Procedures:

1. Make a thin blood film by positioning the cover slip 30º~45º on a drop of blood, and wait it to dry completely.
2. Then, place the slide on the rack and fully cover it with the Leishman stain.
   
   
   
3. Wait for 4 minutes and add few drops of buffer with pH 6.5
   
   
   
4. Gently blow or rock the slide to evenly mix the buffer with the stain.
5. Wait for 12 minutes before washing those stains off from the slide with smooth running water.
6. Allow the slide to be air dried.
7. Look under microscope.

Examples:

The platelets are loosely distributed among the red blood cells.

STart analyser / PT, aPPT & INR

Specimen: Patient's blood within the Sodium Citrate tube ( Blue top )

What is INR?
INR is known as the international normalized ratio.
It is used to monitor patient who has been treated with an anticoagulant, warfarin for blood thinning purpose.
It is a calculation for the appropriate drug dosage for patient based on the result of PT.





*normal=control
*ISI = ISI value for the analytical system being used.






Definition for PT test?
PT test is often used along with APTT to investigate on any bleeding or clotting disorder.

The PT evaluates the coagulation factor VII, X, V, II and I ( Fibrinogen ).
The APTT evaluates coagulation factor XII, XI, IX, VIII, X, V, II (Prothrombin), and I (Fibrinogen) , as well as PK (Prekallikrein) and HK (High Molecular Weigh Kininogen).

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STart analyzer

*The blue pointer show the pipette tips*
*The red pointer shows the finnpipette*

Steel ball inside the cuvette ( pointed )

Methodology:

PT

1. 50µl Plasma is inserted into a cuvette which containing a steel ball, and placed on the channel of the STart analyser.
2. Next, start the timer and incubate for 60sec.
3. After incubation, put the measuring test tube on the rack for the addition of reagent.
4. 100µl of Neoplastine CI plus can be released from the finnpipette only when the release button is pressed.

APTT

1. 50µl Plasma + 50µl CK Prest are inserted into a cuvette which containing a steel ball, and placed on the channel of the STart analyser.
2. Next, start the timer and incubate for 180sec.
3. After incubation, put the measuring test tube on the rack for the addition of reagent.
4. 50µl of CaCl2 (0.025 pre-warmed) reagent is added.

Blood coagulation factors

Blood clotting factors

1. Factor I          - Fibrinogen
2. Factor II         - Prothrombin
3. Factor III       - Thromboplastin
4. Factor IV       - Calcium
5. Factor V         - Proaccelein, Labile Factor
6. Factor VI       - Accelerin
7. Factor VII      - Proconvertin, stable factor
8. Factor VIII     - Antihaemophilic globulin (A.H.G.)
                          - Antihaemophilic factor A (A.H.F.)
9. Factor IX        - Christmas factor,
                           - Antihaemophilic factor B,
                           - Plasma thromboplastin component ( P.T.C.) 
10. Factor X         - Stuart - power factor
11. Factor XI        - Plasma thromboplastin antecedent ( P.T.A.)
12. Factor XII       - Hageman factor
13. Factor XIII      - Fibrin stabilizing factor

Coagulation factors are activated by:

• Foreign surface
• Tissue factor
• Plasma protein factors
• Calcium ions
• Platelets

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Classic blood coagulation theory of Morawitz

Haemostatic mechanism (Haemostasis) can be divided into 2 stages: Initiation stage and Maintenance stage. 

Initiation stage
Initiation factor - prevent bleeding from wound.

• Temporary reflex nervous vasoconstriction of injured vessels.
• Clumping of platelets to plug the injured vessels.
  Those platelets will release serotonin which resulted in constriction of blood vessel adjacent to the wound.
• Tissue tension - The blood escape to the tissue space and causes swelling, thus increase the the tissue tension which further narrow down the surrounding blood vessel.

Maintenance stage

Formation of fibrin clot - involving many coagulation factors.

Complete Blood Count(CBC)

In this laboratory, we have two Coulter LH750 analysers to deal with the FBC test.

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CBC Definition:
Complete blood count or known as Full blood count (FBC), is a panel of tests that used to evaluate 3 types of blood cells.



WBC evaluation : infection, cancer, allergies, inflammation.

• WBC count - total no. of WBC
• WBC Differential - measure the no. of various types of WBC
• Neutrophils
• Eosinophils
• Basophils
• Lymphocytes
• Monocytes

• When the result for WBC differential is not shown on the analyser, it indicates that the patient's sample is either jaundice or lipidemic. 
• To solve the problem, the sample can be diluted and the test is redo once again.
  **Diluent 500μl+sample 500μl** . 
• The result for WBC, RBC, HGB, HCT and platelets should times 2.
• Sometimes, even the sample has been diluted but the differential result is not shown by the analyzer. To overcome this, I need to do the counting manually using microscope.

RBC evaluation

• RBC count - actual no. of RBC
• Hemoglobin - total no. of oxygen-bearing protein in the blood, generally reflects the RBC no.
• Hematocrit - measure the % of a person's total blood volume which containing RBC
• Physical characteristic of RBC:
• MCV
• MCH
• MCHC
• Red cell distribution width (RDW) - measure the variation in the RBC size.
• Reticulocyte count - measure the absolute count / percentage of young RBC.

Platelets evaluation

• Platelets count - no. of platelets
• Mean platelet volume (MPV) - measure the average size of platelets
• Platelet distribution width (PDW) - reflect uniformity of platelets in size. 

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More information about the Explanation of CBC result:

Result explanation of WBC, RBC and Platelets evaluation

WBC Evaluation 

TEST	FULL NAME	EXAMPLES OF CAUSES OF A LOW COUNT	EXAMPLES OF CAUSES OF A HIGH COUNT
WBC	White Blood Cell Count (See reference range)	Known as leukopenia Bone marrow disorders or damage Autoimmune conditions Severe infections (sepsis) Lymphoma or other cancer that spread to the bone marrow Dietary deficiencies Diseases of immune system (e.g., HIV/AIDS)	Known as leukocytosis Infection, most commonly bacterial or viral Inflammation Leukemia, myeloproliferative disorders Allergies, asthma Tissue death (trauma, burns, heart attack) Intense exercise or severe stress
Diff	White Blood Cell Differential (Not always performed; may be done as part of or in follow up to CBC; see reference range)
Neu, PMN, polys	Absolute neutrophil count, % neutrophils (See reference range)	Known as neutropenia Severe, overwhelming infection (sepsis) Autoimmune disorders Dietary deficiencies Reaction to drugs, chemotherapy Immunodeficiency Myelodysplasia Bone marrow damage (e.g., chemotherapy, radiation therapy) Cancer that spreads to the bone marrow	Known as neutrophilia Acute bacterial infections Inflammation Trauma, heart attack, or burns Stress, rigorous exercise Certain leukemias (e.g., chronic myeloid leukemia) Cushing syndrome
Lymph	Absolute lymphocyte count, % lymphocytes (See reference range)	Known as lymphocytopenia Autoimmune disorders (e.g., lupus, rheumatoid arthritis) Infections (e.g., HIV, viral hepatitis, typhoid fever, influenza) Bone marrow damage (e.g., chemotherapy, radiation therapy) Corticosteroids	Known as lymphocytosis Acute viral infections (e.g., chicken pox, cytomegalovirus (CMV),Epstein-Barr virus (EBV), herpes, rubella) Certain bacterial infections (e.g., pertussis (whooping cough),tuberculosis (TB)) Toxoplasmosis Chronic inflammatory disorder (e.g., ulcerative colitis) Lymphocytic leukemia, lymphoma Stress (acute)
Mono	Absolute monocyte count, % monocytes (See reference range)	Usually, one low count is not medically significant. Repeated low counts can indicate: Bone marrow damage or failure Hairy cell leukemia Aplastic anemia	Chronic infections (e.g., tuberculosis, fungal infection) Infection within the heart (bacterial endocarditis) Collagen vascular diseases (e.g., lupus, scleroderma, rheumatoid arthritis, vasculitis) Monocytic or myelomonocytic leukemia (acute or chronic)
Eos	Absolute eosinophil count, % eosinophils (See reference range)	Numbers are normally low in the blood. One or an occasional low number is usually not medically significant	Asthma, allergies such as hay fever Drug reactions Parasitic infections Inflammatory disorders (celiac disease, inflammatory bowel disease) Some cancers, leukemias or lymphomas Addison disease
Baso	Absolute basophil count, % basophils (See reference range)	As with eosinophils, numbers are normally low in the blood; usually not medically significant	Rare allergic reactions (hives, food allergy) Inflammation (rheumatoid arthritis, ulcerative colitis) Some leukemias Uremia

© 2001 - 2015 by American Association for Clinical Chemistry

RBC Evaluation 

TEST	FULL NAME	EXAMPLES OF CAUSES OF LOW RESULT	EXAMPLES OF CAUSES OF HIGH RESULT
RBC	Red Blood Cell Count (See reference range)	Known as anemia Acute or chronic bleeding RBC destruction (e.g., hemolytic anemia, etc.) Nutritional deficiency (e.g., iron deficiency, vitamin B12 or folate deficiency) Bone marrow disorders or damage Chronic inflammatory disease Chronic kidney disease	Known as polycythemia Dehydration Lung (pulmonary) disease Kidney or other tumor that produces excess erythropoietin Smoking Living at high altitude Genetic causes (altered oxygen sensing, abnormality in hemoglobin oxygen release) Polycythemia vera—a rare disease
Hb	Hemoglobin (See reference range)	Usually mirrors RBC results, provides added information	Usually mirrors RBC results
Hct	Hematocrit (See reference range)	Usually mirrors RBC results	Usually mirrors RBC results; most common cause is dehydration
RBC indices
MCV	Mean Corpuscular Volume (See reference range)	Indicates RBCs are smaller than normal (microcytic); caused by iron deficiency anemia or thalassemias, for example.	Indicates RBCs are larger than normal (macrocytic), for example in anemia caused by vitamin B12 or folate deficiency, myelodysplasia, liver disease, hypothyroidism
MCH	Mean Corpuscular Hemoglobin (See reference range)	Mirrors MCV results; small red cells would have a lower value.	Mirrors MCV results; macrocytic RBCs are large so tend to have a higher MCH.
MCHC	Mean Corpuscular Hemoglobin Concentration (See reference range)	May be low when MCV is low; decreased MCHC values (hypochromia) are seen in conditions such as iron deficiency anemia and thalassemia.	Increased MCHC values (hyperchromia) are seen in conditions where the hemoglobin is more concentrated inside the red cells, such as autoimmune hemolytic anemia, in burn patients, and hereditary spherocytosis, a rare congenital disorder.
RDW (Not always reported)	RBC Distribution Width	Low value indicates uniformity in size of RBCs.	Indicates mixed population of small and large RBCs; young RBCs tend to be larger. For example, in iron deficiency anemia or pernicious anemia, there is high variation (anisocytosis) in RBC size (along with variation in shape – poikilocytosis), causing an increase in the RDW.
Reticulocyte Count (Not always done)	Reticulocytes (absolute count or %) (See reference range)	In the setting of anemia, a low reticulocyte count indicates a condition is affecting the production of red blood cells, such as bone marrow disorder or damage, or a nutritional deficiency (iron, B12 or folate).	In the setting of anemia, a high reticulocyte count generally indicates peripheral cause, such as bleeding orhemolysis, or response to treatment (e.g., iron supplementation for iron deficiency anemia).

© 2001 - 2015 by American Association for Clinical Chemistry

Platelet Evaluation 

TEST	FULL NAME	EXAMPLES OF CAUSES OF LOW RESULT	EXAMPLES OF CAUSES OF HIGH RESULT
Plt	Platelet Count (Seereference range)	Known as thrombocytopenia: Viral infection (mononucleosis, measles, hepatitis) Rocky mountain spotted fever Platelet autoantibody Drugs (acetaminophen, quinidine, sulfa drugs) Cirrhosis Autoimmune disorders Sepsis Leukemia, lymphoma Myelodysplasia Chemo or radiation therapy	Know as thrombocytosis: Cancer (lung, gastrointestinal, breast, ovarian, lymphoma) Rheumatoid arthritis, inflammatory bowel disease, lupus Iron deficiency anemia Hemolytic anemia Myeloproliferative disorder (e.g., essential thrombocythemia)
MPV (Not always reported)	Mean Platelet Volume	Indicates average size of platelets is small; older platelets are generally smaller than younger ones and a low MPV may mean that a condition is affecting the production of platelets by the bone marrow.	Indicates a high number of larger, younger platelets in the blood; this may be due to the bone marrow producing and releasing platelets rapidly into circulation.
PDW (Not always reported)	Platelet Distribution Width	Indicates uniformity in size of platelets	Indicates increased variation in the size of the platelets, which may mean that a condition is present that is affecting platelets

© 2001 - 2015 by American Association for Clinical Chemistry

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Reference:

1. Labtestsonline.org,. 'Complete Blood Count: The Test | Complete Blood Count: CBC Test | Lab Tests Online; Complete Blood Count Test: CBC; Hemogram; CBC With Differential | Lab Tests Online'. N.p., 2015. Web. 5 Nov. 2015.

HbA1c test

Definition of HbA1c test:

• Hemoglobin A1c test, is an important in vitro diagnostic test for quantitative determination of glycated hemoglobin in human whole blood.
• It aids in the diagnosis of diabetes mellitus.
• A1c (glycated hemoglobin) is formed in the blood when glucose attached on the hemolobin.

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Procedure:
(*Everyday start with QC)

1. Shake tube and record patient's data in the book.
2. Take cartridge ( Make it stand on the table, because inside have solution)
   (*Do not touch or leave any fingerprint on the bottom part of the cartridge)
3. Press the "patient" icon & insert the cartridge
4. Insert user ID and patient MRN.

Cartridge

AS100 analyser.

Cartridge ( Before )
Have to use the capillary tube to obtain the required amount of blood for testing.

 

Cartridge ( After )

Cartridge ( After ) which the capillary tube is shown **the blood sample was replaced with blue reagent.**

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HbA1c levels:

• Normal:           4% & 5.6%
• Abnormal:       5.7% & 6.4%   (39 & 47 nmol/mol)

Limitations:

(Cannot diagnose)

• Patient with a hemoglobinopathy
• Patient with abnormal red cell turnover
• Patient with iron deficiency & hemolytic anemia,...
• Patient that have received a blood transfusion/ cancer chemotheraphy within the past 3 weeks
• Cannot use coagulated/ hemolysed sample

ESR count with Wintrobe's method

What is ESR ( Erythrocyte sedimentation rate )?
ESR is the rate at which the cells settle. If the blood to which an anticoagulant has been added is allowed to stand vertically in a tube for 1 hour, the cells will settle to the bottom, leaving the plasma as a clear supernatant fluid.

Procedures:

1. Fill the blood in the tube until it reach the line, then fully mix the blood with the anticoagulant by inversion.
2. Insert the capillary tube and allow it to stand vertically for 1 hour. ( write down the time and start the timer)  

•   Compare  with the HGB ( Hemoglobin ) result. Normally if the HGB result is normal,the blood cells will sediment very slow.

Factors that affect the sedimentation:

• Rouleaux formation
   
  ( RBC may form aggregates in the presence of plasma protein or other macromolecules )
  
• Plasma content of blood
• Fibrinogen ↑ ESR ↑  , (Fibrin will control the rouleaux formation)
• Globulin     ( Reduction of globulin will indicate the red cell's sedimentation rate )
• Albumin↑ ESR↓
• Ratio of cells to plasma  ( ↓no. of blood cells favor the formation  of rouleaux,↑ ESR )
• Deviation of the tube from the vertical position,↑ ESR
• Temperature

Precautions:

• Slanting of tube
• Inclusion of air bubbles in the blood column
• Leakage from the bottom of tube

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Reason of rapid sedimentation rate:

1. Extensive inflammation, cell destruction, toxemia.
2. Pregnancy ( after second month )
3. Menstruation
4. Acute myocardial infarction
5. Infected, necrotic/maglinant tumor
6. Liver disease
7. Tuberculosis ( esp. Miliary tuberculosis) 
8. Rheumatic fever
9. Active rheumatoid arthritis
10. Nephrosis ( low blood albumin, anaemia )
11. Shock
12. Active syphilis ( moderate acceleration )
13. Postoperative states
14. Active infectious disease
15. Salpingitis, appendicitis 
16. Puerperium (the time from the delivery of the placenta through the first few weeks after the delivery.)

Reason of Slow sedimentation rate:

1. Newborn infant
2. Polycythemia
3. Congestive heart failure
4. Allergic condition
5. Sickle cell anemia

Red Blood Cells Indices

Abbreviations:

RBC --- Red Blood Cell
Hb --- Hemoglobin
P.C.V --- Packed cells volume / Hematocrit

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Mean Cell Volumn / Mean Corpuscular Value

Definition: Average volume/size of a single RBC.




Normal Range: 76~96 u3

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Mean Cell Haemoglobin / Mean Corpuscular Haemoglobin

Definition: Average hemoglobin content of a single RBC / Average weigh of Hb in the RBC.



Normal Range: 27~32 uug

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Mean Cell Haemagoblin concentration / Mean Corpuscular Haemagoblin concentration 

Definition: Average concentration of Hb in the RBC volume.



Normal Range: 32%~36%

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References:

1. Labce.com,. 'Red Blood Cell Indices: Definitions And Calculations - Labce.Com, Laboratory Continuing Education'. Web. 3 Nov. 2015.