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The order of collection for microcollection tubes:

CBGs

Blood smear

Lavender EDTA tubes

Green, light green, amber lithium heparin tubes

Gray EDTA tubes

Gold, amber, red serum tubes

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In fully automated analyzers that use spectrophotometry, samples left standing for too long can produce falsely elevated values.

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an increase in band cell is known as left shift

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3 Stages of Erythrocyte Sedimentation:

i. Stage 1: 10 mins – lag/aggregation phase . Initial stage where rbcs stack one another like coins, a process called "Rouleaux formation". This process can be elevated by certain proteins in the plasma that disrupt the zeta potential between the cells like immunoglobulins, fibrinogen, and othrr acute phase reactants.

ii. Stage 2: 40 mins – decantation phase. The stacks of rbcs begin to settle at the bottom of the tube.

iii. Stage 3: 10 mins – packing phase. As more stacks of rbcs settle at the bottom, they pack together and slow down the sedimentation process until it stops.

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Never Let Monkey 🐒 Eat Banana 🍌

From most to least abundant WBC in the PB

Neutrophil - 50-70%

Lymphocyte - 18-42%

Monocyte - 2-11%

Eosinophil - 1-3%

Basophil - 0-2%

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Using EDTA as an anticoagulant may cause platelet satellitism, leading to falsely low platelet counts and falsely high white blood cell counts . To prevent this, a sodium citrate tube can be used.

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Nucleus-cytoplasm asynchrony refers to the incongruent development of the nucleus and cytoplasm to each other during erythropoiesis. It is notably present in megaloblastic anemia particularly when there is a deficiency in the patient's folate and vitamin b12 levels. The nucleus matures slower than the cytoplasm, since the maturation of the nucleus is highly dependent on these nutrients.

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Updated Myeloid to Erythroid Ratio = 1.5:1 to 3.3:1

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Decreased ESR:

Sickle Cell & Sphreocytes

Cold Temperature

More anticoagulant than RBC

Increased ESR:

Menstruation

Inflammation

Warm Temperature

Rheumatoid Arthritis

Incorrect Anticoagulant

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De Jesus, Adrian Delfin L.

Volume Distribution sub groups histograms

35-90 fL= LYMPHOCYTES

90-160 fL= MONONUCLEARS

160-450 fL= GRANULOCYTES

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Anisocytosis is the variation in the size of the RBC. Poikilocytosis is the variation in shape of the RBC. Anisochromia is the variation in Hgb concentration or Central Pallor

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The dilution used in RBC count is 1:200 when blood is aspirated up to 0.5 mark of Thoma pipette

- Darlene Vitancol

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Always observe proper decorum in the workplace. Pure intelligence can’t take you anywhere.

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Proper anticoagulant use and correct blood-to-additive ratio are essential to avoid clotting or dilution errors that can affect test results.

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Parazo, Ma. Liezl C.

RBC parameters include: MCV, MCH, MCHC, and RDW. These are important for diagnosing conditions, particularly in differentiating or identifying anemias.

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When determining the RBC count using 2 chambers, always check first if the difference of the count between the 2 chambers is within the allowable range (10%). If it is below 10%, then we can proceed with getting the average of the total cells counted in both chambers as the cells counted to be inputted in the main formula.

cell counts = [(average cell count from 2 chambers) (DF)]/[(area)(depth)]

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Firmacion, Shane Marie I.

Forward scatter (0 degree) is for cell volume

Side scatter (90 degrees) is for cell granularity

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In blood extraction, it is important to make sure that the patient is not diabetic before considering extraction in the veins of the foot.

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WHO is the organization that is widely used to classify leukemia. It is now the standard classification in diagnosing leukemia. It subdivide leukemia according to:

1. Cellular morphology

2. Cytochemical stains

3. Immunophenotyping

4. Cytogenetics abnormalities

5. Clinical Syndrome

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Protein buildup in automated machines using electrical impedance causes falsely increased cell volume and falsely decreased cell count.

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Normal MCV, MCH, MCHC

Microcytic, Hypochromic = SCHILT

Sideroblastic Anemia

Anemia of Chronic Disease/Chronic

Inflammation

Hemoglobin E Disease/Trait

Iron Deficiency Anemia

Lead Poisoning

Thalassemia

High MCV, Normal MCHC

Macrocytic, Normochromic = Megaloblastic and Non megaloblastic Anemia

Low MCV, Low MCHC

Normocytic, Normochromic = Hemolytic Anemia

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Always label the specimen with key patient details such as name, age, date and time of collection, and birthdate. An accurate patient identification is essential to prevent misidentification and mislabeled specimens, which can lead to misdiagnosis.

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SLS = Sysmex

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esterases that differentiates AML (+)

1. Napthol - ASD Chloroadetate esterase

2. Napthyl Acetate esterase

3. Napthyl butyrate

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Electrical impedance can detect electrical resistance produced by a cell; however, it is limited by the ability to only distinguish cells by their volume.

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ESR can falsely increase in the following situations: usage of potassium oxalate, increase or high room temperature, inflammatory conditions, severe anemia, and unstable platform.

Whereas falsely decreased ESR occurs when it is unprocessed for more than four hours, increase usage of anticoagulant, and lower room temperature.

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electronic impedance cannot identify specific granules

fluorescent stains tend to appear stronger in immature cells

-Meann Basco (sec6)

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The principle of electrical impedance is the measurement of changes in electrical resistance produced by cells as they traverse a small aperture.

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The dilution used in RBC count is 1:200 when blood is aspirated up to 0.5 mark of Thoma pipette

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the pH of Sodium Phosphate buffer used for Wright-Giemsa stain is 6.4.

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Heparinized blood is not used for hematologic tests stained with Wright's stain, such as CBC, because the smear will become too blue.

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A shift to the left indicates an increase in young forms of WBCs, while a shift to the right indicates an increase in mature forms.

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Fe2+ (Ferrous) is the reduced form of iron which is absorbable by the body. On the other hand, Fe3+ (Ferric) is the oxidized form that cannot be absorbed by the body.

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Reticulocyte count is a sensitive measure of RBC production which assess the erythropoietic activity of the bone

marrow

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Isovolumetric sphering is done to prevent the bending and deforming of RBCs

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When there is more anticoagulant than blood the ESR is decreased. Anticoagulants can affect the electrical charge on RBC membranes, increasing repulsion between cells. This prevents aggregation, which slows sedimentation.

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Rule of 3:

Hgb x 3 = Hct +- 3

Rbc x 3 = Hgb +- 0.5

Useful for checking the accuracy of automated methods against manual methods quickly

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Rule of Three is used to check the validity of results. It only works on NORMOCYTIC, NORMOCHROMIC specimens

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Platelet Count Diluting Fluid: 1% Ammonium Oxalate

Dermal Puncture Incision depth-

Infants: 0.35-1.6 mm

Adults: 3 mm

(Both below the skin)

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Males have a higher reference value for RBC count because of their testosterone levels.

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Sodium lauryl sulfate is a widely used cyanide-free reagent

Antonio, Eldwin R.

Sec 8

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Venipuncture using winged blood collection set requires a discard tube

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Specimen collection is the first and most critical step in laboratory testing thus proper technique ensures accurate results and patient safety. Mistakes in collection can lead to false results or the need to recollect.

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Reference ranges

RBC: 4.2 - 6x10^12/L (male), 3.80 - 5.20x10^12/L (female)

HGB: 13.5 - 18 g/dl (male), 12 - 16 g/dl (female)

HCT: 40-54% (male), 36-47% (female)

MCV: 80-100 fL

MCH: 26-34 pg

MCHC: 32-36 g/dL

Retics: 20-115x10^9/L

WBC: 3.6-10.6x10^9/L

NEUT: 50-70%

LYMPH: 18-42%

MONO: 2-11%

EO: 1-3%

BASO: 0-2%

PLT: 150-450x10^9/L

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Cytochemical staining

specific for proteins, enzymes, found in granules specific for WBC.

Eosinophil: IL-3, IL-5, G-CSF

for growth and survival: IL-3 and IL-33

BM and SPLEEN: IL-3

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Tests affected by anticoagulant/additive contamination: (PECH)

Potassium oxalate

EDTA

Clot activator / Silica

Heparin

Lachica, Angel Ann T. (Sec 5)

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In calculating corrected hemoglobin value, a PLASMA HEMOGLOBIN can be used as a blank. -Reina Lim (Sec 8)

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Bending, deforming, misaligning of RBCs can be prevented by isovolumetric sphering.

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Osmotic fragility testing is performed to detect the presence of spherocytes.

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MICROCYTIC HYPOCHROMIC ANEMIA:

T =thalassemia

A = anemia of chronic inflammation

I = iron deficiency anemia

L = lead poisoning

S = sideroblastic anemia

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Wright stain - polychromatic erythrocyte

Supravital Stain - reticulocytes

Examples of supravital stain

New Methylene Blue

Brilliant Cresyl Blue

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In Fluorescent staining of reticulocytes and platelets, The more immature the cell, the higher the intensity of the fluorescent dyes.

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In the cyanmethemoglobin method, potassium ferricyanide oxidized Hemoglobin to methemoglobin and potassium cyanide turns it to cyanmethemoglobin.

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A long-standing, unmixed EDTA blood sample can lead to falsely elevated hemoglobin results due to the settling of red blood cells (RBCs) in the tube. Automated hematology analyzers, especially those with probes that aspirate from the bottom, will encounter a higher concentration of RBCs in the unmixed sample, resulting in a falsely increased hemoglobin reading.

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The diluting fluid used in RBC count is an isotonic solution (e.g. NSS) because it matches the osmolality of the blood, which ensures that the red blood cells maintain their natural morphology during the counting process.

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In preparing blood smears for reticulocyte count, always remember that a 1:1 ratio of EDTA whole blood and New Methylene Blue is mixed and should be left undisturbed for 3-10 minutes before smearing.

Alex Aguerra

Sec 8

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Alternative reagent for determining hemoglobin is Sodium Lauryl Sulfate

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Direct Current Impedance measures the size of a cell while Radio Frequency Resistance measures the complexity or granularity of a cell.

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RBC diluting fluid

1. Hayer’s

2. Dacie

3. 1% isotonic saline

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In fluorescent staining, the more immature the cell, the greater is the intensity of fluorescent staining.

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Jennuel Herbert B. Frias

Macrocytic, Normochromic

• Megaloblastic Anemia due to Vit B12 or Folate deficiency

• Pernicious Anemia

Normocytic, Normochromic

• Hemolytic Anemia

• Sickle Cell anemia

Microcytic, Hypochromic

• Thalassemia

• Anemia of Chronic Disease

• Iron deficiency anemia

• Lead poisoning

• Sideroblastic anemia

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LIM, JOAN MARI M.

The use of a discard tube is necessary when using a butterfly winged infusion set for venipuncture. A 0.5mL volume of blood does not enter the first tube and fills the plastic tubing instead, interfering with coagulation tests.

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Sodium Lauryl Sulfate (SLS) is an alternative reagent used in hematology analyzers for hemoglobin determination

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Sodium Lauryl Sulfate is an alternative reagent for hemoglobin determination

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Forward-angle light scatter (0 degrees) correlates with cell volume, while side light scatter (90 degrees) correlates with degree of internal complexity.

-Monje, Jemima Grace D. (Sec 6)

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basophilic stippling is seen in lead poisoning

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Charcot-leyden crystal is utilized as a marker to identify if the cell is from the eosinophilic lineage

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Cytochemical staining: usually detects presence proteins or enzyme, help identify specific cells (ex. charcot leyden crystals= eosinophil)

reactions: may impart different colors

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In reticulocytes and platelets, the more immature the cell, the greater the intensity of fluorescent staining.

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Drabkin’s reagent is composed of potassium ferricyanide, potassium cyanide, sodium bicarbonate and surfactant.

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Specimen collection is the process of gathering samples (like blood, urine, stool, sputum) for laboratory testing. It must be done correctly to ensure accurate results.

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Rule of 3 is only applied in normocytic normochromic anemia

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If the pH of a smear is too acidic, the RBCs appear bright red and the nuclei of the WBCs appears pale blue. And if it is too basic, the RBCs will appear deep blue and WBC structures are hardly discerned.

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M:E ratio

1.5:1 - 3.3:1

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Sulfhemoglobin cannot be converted to cyanmethemoglobin and cannot be measured using the cyanmethemoglobin method (reference method approved by CLSI for hemoglobin determination)

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Erythrocyte Sedimentation Rate refers to the speed of settling of RBC in anticoagulant blood.

-Borje, Myrrh Yngryd

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Reference value for reticulocytes % adults: 0.5-2.5% and neonates: 1.5 to 6%

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The use of Sodium Lauryl Sulfate as an alternative in hemoglobin determination (cyanmethemoglobin method) lessens the use cyanide

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In fluorescent staining, the more immature cells, the greater the intensity

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Microcytic Hypochromic Anemia:

S: Sideroblastic Anemia

C: anemia of Chronic Disease

H: HBe Disease/Trait

I: Iron Deficiency anemia

L: Lead Poisoning

T: Thalassemia

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IL-3 and IL-5 are responsible for differentiation of eosinophils. IL-3 and IL-33 are responsible for eosinophil survival

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In a peripheral blood smear, the film is 2/3 to 3/4 the length of the slide and the film should be finger shaped, very slightly rounded at the feather edge, not bullet shaped.

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The light blue top (sodium citrate) should be filled exactly, neither underfilled nor overfilled, to avoid erroneous results in coagulation tests.

PLACINO, ERICA D.

Sec 8

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DIFFERENCE BETWEEN HISTOGRAMS VS. SCATTERPLOTS

Histograms focus only on the cells number and volume. Scattergrams focus on 2 or more cell properties plotted against each other (e.g., size vs. granularity/lobularity)

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Isotonic solution is the diluting fluid used for RBC count because it matches the osmolality of the blood and therefore, will not affect the morphology of the RBC.

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An increased amount of Band Cells may be due to pregnancy and a strenuous exercise.

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Eosin stain - 5 dips

Methylene blue stain - 25 dips

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the reference method for measuring hemoglobin is cyanmethemoglobin method

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In counting cells in the Neubauer Chamber, it is important to always count from two sides of a grid also known as the “Inverted L Rule.” ❤️‍🔥

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David, John Kieffer M.

Sec 8

Reticulocyte Count uses supravital stains (e.g. new methylene blue) to visualize RNA remnants.

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For optical light scatter, the light source can either be tungsten-halogen lamp or helium-neon laser.

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Osmotic fragility is decreased when sickle cells and target cells are present and is increased in the presence of spherocytes.

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Manual RBC counts are rarely performed because of the inaccuracy of the count and questionable necessity.

-Asuncion, Sec 6

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Stages of ESR

First 10 mins: rouleaux

40 mins: sedimentation

Last 10 mins: stage packing

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Median cubital vein is the vein of choice (1st choice) for venipuncture.

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Proper patient identification is the first and most crucial step in the venipuncture procedure

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Cyanmethemoglobin method is the reference method for measuring hemoglobin. Also, sodium lauryl sulfate is a commonly used cyanide-free reagent for most hematology analyzers.

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Sulfhemoglobin is not quantitated in the cyanmethemoglobin method as it isn’t converted to cyanmethemoglobin

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Cyanmethemoglobin (Hemiglobincyanide) Method is the reference method for hemoglobin determination using the Drabkin's reagent.