ROUTINE HEMATOLOGY PROCEDURES

Hemoglobin Measurement

Hematocrit Measurement

Blood Cell Counts (RBC, WBC)

Preparation and Staining for Blood Smears

Erythrocyte Sedimentation Rate

Reticulocyte Count

Eosinophil Count

Sickle Cell Tests

HEMOGLOBIN MEASUREMENT

HEMOGLOBIN MEASUREMENT HEMOGLOBIN: main component of the RBC that

serves as the vehicle for the transportation of oxygen and carbon dioxide.

- It imparts red color to the blood.

- It buffers blood pH.

Purpose of estimating hemoglobin To detect the oxygen carrying

capacity of blood.

The result assists in detecting diseases, which causes a deficiency or excess of hemoglobin.

Studying changes in hemoglobin concentration before or after operations and blood transfusions.

To detect anemia and its severity and to monitor an anemic patients response to treatment.

To check hemoglobin level of blood prior to donating blood.

To calculate red cell indices.

Normal Values

The normal value depends on the age and sex of the individuals:

MALE: 13-18 g/dl FEMALE: 12-16 g/dl AT BIRTH: 15-20 g/dl

Methods of Estimation of Hemoglobin

Colour based (Colorimetric): Based on the colour of hemoglobin or a derivative of hemoglobin.

Physical method (Gravimetric): Based on specific gravity.

Chemical method: Based on iron content of hemoglobin.

Gasometric method: Based on oxygen combining capacity of hemoglobin.

Spectrophotometric method: Based on measurements using spectrophotometric devices.

COLORIMETRIC METHOD:1. Sahli’s Method or Acid Hematin method

Principle

Hemoglobin is converted to acid hematin by N/10 HCl, the resulting brown colour is compared with standard brown glass reference blocks.

The intensity of the brown colour depends on the amount of acid haematin produced, which in turn depends on the amount of hemoglobin in the blood sample.

Reagents:

N/10 Hydrochloric acid (HCl)

Distilled water for dilution.

Blood anticoagulated with EDTA

N/10

Procedure

Place N/10 HCl in the tube up to the lowest mark.

Draw blood up to 20 mm mark in the pipette and transfer it to the acid in the tube.

Rinse the pipette well by drawing up the acid and re expressing it. Mix the acid and blood by shaking the tube well.

Allow it to stand for at least 10 minutes - to allow brown colour to develop due to the formation of acid hematin.

Now dilute the solution with distilled water drop by drop with continuous mixing, using the glass rod provided.

Procedure (contd....) Match the color with

that of the glass plates in the comparator.

Reading is taken when the color of the solution in the tube exactly matches the comparator. Matching should be done at eye level against natural light.

The level of the fluid at its lower meniscus is noted and the reading on the scale corresponding to this level is read as gram/dl.

Sources of Errors

Technical errors

Improper mixing of blood,

Errors in pipetting,

Tissue fluid contaminating capillary blood.

Visual errors – Taking the reading is very subjective, as it is a comparison of colours. It can vary from person to person. So the results may not be accurate.

Quality of the color comparators can affect the reading – If the glass blocks are old or faded it can cause wrong results.

Insufficient time allowed for the conversion of Hb to acid haematin.

A minimum of 10 minutes is required for the reaction to be almost complete, otherwise biological false negative result is obtained.

Carboxyhemoglobin, methemoglobin and sulfhemoglobin are not converted to acid haematin.

Non-hemoglobin substances such as protein, liquid and cell stroma interfere with the colour of blood diluted with acid and hence give false results.

Time delay - The brown colour of acid haematin is not stable, so undue delay in reading the test result is not allowed.

2. Alkaline Hematin method

In this method the Hb is converted to alkali hematin by the addition of N/10 NaOH.

The alkali hematin gives a brown colour that can be read against comparator standards or in a colorimeter.

Apparatus: Photo electric meter with green filter. N/10 NAOH 0.05 ml pipette Standard (Gibson’s and Harrison’s): This is a

mixture of chromium potassium sulphate, cobaltous sulphate and potassium dichromate in aqueous solution. The solution is equal in colour to 1 in 100 dilution of blood containing 16.0 Hb per dl.

Technique: Add 0.05 ml of blood to 4.95 ml of N/10 NAOH.

Mix well and boil for 4 minutes, along with 5 ml standard solution.

Cool quickly in cold water,and match the test against standard using colorimeter using green filter.If the test give too high value add 5.0 ml of water and read again.

Advantage 1) Unlike Sahli’s method, carboxyhemoglobin,

sulfhemoglobin are converted to alkali hematin.

2) Fetal haemoglobin is resistant to denaturation by alkali and this method is used to determine the level of fetal haemoglobin in blood.

Disadvantage The solution of Hb in alkali has to be heated to

ensure complete denaturation.

Note:

Matching should be done within 30 minutes after boiling

Spectrophotometric method

The esimation is based on Beer’s and Lambert’s law ( OD= HGB CONC)

1) Cyanmethemoglobin method

2) Oxyhemoglobin method

Cyanmethaemoglobin method

This is the preferred and the most accurate method for determining the hemoglobin concentration in laboratory.

Principle: ***Blood is diluted in a solution of

potassium ferri cyanide and

potassium cyanide. ***The absorbance of the solution is then measured in a spectrophotometer at a wavelength of 540 nm or in a colorimeter using a yellow-green filter.

Reagents Hemoglobincyanide standard Detergent modified Drabkin’s

solution

Potassium Ferricyanide

Potassium Cyanide

Potassium Dihydrogen Phosphate

(ORIGINALLY, NaHCO3)

Non-ionic detergent/Sterox SE/Triton X-100/Saponin

Distilled water

Procedure

Take 5ml of Drabkin’s solution in a large sized test tube.

Add 20 micro litres of well mixed anticoagulated venous blood.

Rinse the pipette and mix well.

Allow it to stand at room temperature for 3 - 10 minutes.

Absorbance is measured against reagent blank at 540 nm either in a spectrophotometer or in colorimeter.

Advantages All forms of Hb except SHb are readily

converted to HiCN. Direct comparison with HiCN standard

possible. Stability of the diluted sample. Easy to perform the test. Reagents are readily available. The standard is stable.

Disadvantages Potassium cyanide in the solutions is poisonous,

though it is present only in a very low concentration hence the reagents should be handled carefully.

Explosion can occur if undiluted reagents are poured in the sink. Hydrogen cyanide is released by acidification and the gas if it accumulates can result in explosion. Reagents and samples should be disposed along with the running water in the sink.

Increased absorbance not due to hemoglobin may be caused by turbidity due to abnormal plasma proteins, hyperlipemia, high WBC count or fat droplets, and the presence of HbS and Hb C.

FALSE CYANMETHEMOGLOBIN MTD:

presence of turbidity due to.. HIGH WBC COUNT: to correct,

CENTRIFUGE

READ SUPERNATANT HbS and HbC: to correct, DILUTE 1:1 with

H2O result x 2

Lipemic blood: to correct, PREPARE PATIENT’S BLANK (patient plasma + HiCN reagent)

Specific gravity method (Physical method)

Assesses hemoglobin concentration via blood specific gravity.

Mass screening SG of CuSO4: 1.053 (equivalent to

12.5g/dL)

PROCEDURE:

1.Collect blood sample

2.Drop a blood the copper sulfate solution (distance: 1 cm)

3.Observe the activity of the blood

INTERPRETATION: (Within 15 SECONDS describe how the drop of blood behaves in the solution)

MAINTAIN: hgb concentration equals to 12.5 g/dL/13.5 g/dL

FLOAT: <1.053 OR <12.5 g/dL

SINK: >1.053 OR >12.5 g/dL

Chemical methods (estimation of the iron content) The principle is based on the fact that each

molecule of haemoglobin contains 4 atoms of iron or 3.47 grams of hemoglobin.

The iron present is detached from the hemoglobin and measured.

The hemoglobin is calculated by using the formula.

Hb(gm/dl) = Blood iron content in mg/dl blood

3.47

Gasometric methods(Measurement of Oxygen combining capacity)

It is done by using van Slyke apparatus.

The principle is based on the fact that one molecule of O2 binds to each iron atom.

So one molecule of hemoglobin binds 4 molecules of oxygen. Thus oxygen combining capacity thus indirectly measures the amount of Hb.

It is estimated that 1 gram of hemoglobin binds about 1.34 ml of oxygen.

From this the haemoglobin concentration is calculated by using the following formula.

Hb in gm/dl = O2 binding capacity in ml/dl blood

1.34

Quality Control

The important aspect of quality control is to identify those steps in which the likelihood of error is high and to consider ways to minimize that likelihood. Some of the measures followed are:

Duplicating samples.

Hemolysate of known value are run with batches of tests.

Haemoglobin values are compared with other values. For example PCV = 3 x Hb. This is true unless there is marked microcytosis or macrocytosis.

 

If haemoglobin values are abnormal either too low or high, check peripheral smear to look for other associated abnormalities.

RULE OF THREE

3 X RBC= HEMOGLOBIN

3 X HGB= HEMATOCRIT 3

Apply only to normocytic, normochromic red cells

HEMATOCRIT MEASUREMENT

HEMATOCRIT MEASUREMENT (Packed Cell Volume)

The percentage of the total volume of WB that is occupied by pRBCs when a known volume of blood is centrifuged at a constant speed for a constant period of time.

Relevance:

1. MOST accurate and simplest for detecting presence and degree of anemia or polycythemia.

2. Essential in determination of red cell indices that help in detecting and classifying various types of anemia.

Principle:

GRAVITY/CENTRIFUGAL FORCE:Due to gravity and centrifugal

force, the dense particle settles down.

1. Macromethod CENTRIFUGE: 2000-2300 for 30mins

1st layer: FATTY LAYER: barely visible unless patient is lipemic

2nd layer: PLASMA

3rd layer: BUFFY COAT (1mm: 10,000 WBCs/mm3)

Bottom: PACKED CELLS

WINTROBE TUBE:

Length: 115 mm LEFT RIGHT

Bore: 3 cm 0-100 100-0

ESR HCT

2. Micromethod *Capillary tube: LENGTH: 70-75 mm

BORE: 1 mm

Holds app. 0.05 ml of WB/ 50 uL of blood

 

*Centrifuge: 10,000-15,000 for 5 mins

Top: PLASMA

2nd: PLATELETS

3rd: LEUKOCYTES

4th: RETICS, nRBCs

5th: MATURE RBCs

BOTTOM: Clayseal (4-6 mm)

RESULTS

Height of packed red cells (mm)

Hematocrit = ------------------------------------------ × 100

Height of packed RBCs and plasma

(i.e, height of blood column)

HCt. = 45/100 × 100 = 45 percent

This means out of 100 volumes or parts ,45 volumes are red cells and 55 volumes are plasma.

3. AUTOMATED METHODS

HCT is COMPUTED.

HCT= MCV X RBC COUNT

Increased PCV

Polycythemia

-Newborns, High altitude, Hypoxia due to lung and heart diseases.

Congestive Heart failure, Burns (loss of plasma), Dehydration, Severe Exercise, Emotional stress

Decreased PCV

All types of Anaemia

Pregnancy (Hemodilution)

Ingestion of large amount of water

BLOOD CELL COUNTS

BLOOD CELL COUNTS

Counting Chambers

1. FUCH’S ROSENTHAL: 4 X 4 X 2

2. SPEIR’S LEVY: 2 X 5 X 4

3. IMPROVED NEUBAUER: 3 X 3 X 2

Red Cell and White Cell Counts

RBC problem: anemia, polycythemia WBC problem: leukocytosis,

leukopenia RBC reference ranges

• Male 4.50 - 6.00 x 106/L (x 1012/L)

• Female 4.0 -5.0 x 106/L (x 1012/L)

WBC reference range• Both 4.5 – 11.0 x 103/L (x 109/L)

DIFFERENCES BETWEEN RBC AND WBC PIPETTE

RBC pipette WBC pipette1) It has a red bead It has a white bead

2) It has graduations up to mark 101

It has graduations up to mark 11

3) Size of bulb is larger Size of bulb is smaller

4) Size of lumen is smaller

Size of lumen is larger

RBC PIPETTE

WBC PIPETTE

Thoma PipetRBC

Aspirate to 0.5 or 1.0

Dilute to 101DF = 200 or

100

WBCAspirate to

0.5 or 1.0Dilute to 11DF = 20 or

10

IMPROVED NEUBAUER HEMOCYTOMETER

Each scale is 3mm wide and 3mm long.

Depth of the chamber is 0.1mm

The whole scale is divided into 9 big squares.

Each square is 1mm long and 1mm wide.

The four corner squares are

further divided into sixteen

smaller squares and are used

for WBC counting.

Four corner squares are

meant for WBC counting.Total = 64

small squares

W

W

W

W

•Central square is divided into 25 medium sized square and are separated by triple line•The medium sized square are further divided into 16 small square(tiny)•The four corner and central square are used for platelet and RBC count.

Counting Rule

Do not count cells touching

Bottom lineRight line This is to avoid

double counting.

Principle

Dilution of blood Sampling of diluted suspension into

measured volume Counting of cell in that volume

FOCUSING

4X to see the general formation of slide.

10X for WBC counting 40X for RBC/Plt. counting

Source of error

False high count False low count Improper mixing

Uneven distribution of cell

Error in pipetting

Error in calculation

Blood taken from area of hemo concentration

Yeast, dirt and leucocyte are counted as RBC

Blood diluted with tissue fluid

Undue delay in counting of cell

Clumping of cell(AIHA)

Uneven distribution of cell

Faulty technique of counting

Improperly standarized counting chamber

*RBC COUNT @ HPO

RBC diluting fluids are ISOTONIC solutions. DILUTION (RBC pipette): 0.5:100 (blood: diluent) =

1:200 RBC count: RBC/mm3= #RBC x Area Correction

Factor (ACF) x Depth Correction Factor (DCF) X Dilution Factor (DF)

= #RBC X 5 X 10 X 200

= RBC X 10,000

SI: x 0.001

RBC Example… Aspirate blood to 0.5, then dilute to 101 DF = 200 Counted 500 erythrocytes in 5 small squares

RBC count: RBC/mm3= #RBC x Area Correction Factor (ACF) x Depth Correction Factor (DCF) X Dilution Factor (DF)

RBC = 500 rbc x 5 x 10 x 200

= 5.00 x 106/mm3 (L)

*WBC COUNT @ LPO WBC diluting fluids are HYPOTONIC SOLUTIONS to

lyse non-nucleated RBCs. Mix for 3 minutes to allow lysis of RBCs. DILUTION: 0.5:10 (Blood:Diluent)= 1:20

*Leukocytosis: Use RBC pipette (1:100 or 1:200) WBC count: WBC/mm3= #WBC x AF x

DCF X DF

= #WBC X 0.25 X 10 X 20

= WBC X 50

SI: x 0.001

WBC Example…

Aspirate blood to 0.5, then dilute to 11 DF = 20 Counted 160 leukocytes in 4 large

squares

WBC/mm3= #WBC x AF x DCF X DF

= #WBC X 0.25 X 10 X 20

= 160 x 0.25 x 10 x 20

= 8.0 x 103/mm3 (L)

** NUCLEATED RBCs are not lysed by WBC diluents. They are then falsely counted as WBCs.

NV: ADULT: ≥5 nRBCs/100 WBC differential

NEWBORN: ≥10 nRBCs/100 WBC differential

CORRECTED WBC COUNT=

PREPARATION AND STAINING FOR BLOOD SMEARS

Types of blood smears: 1. The cover glass smear.

2. The wedge smear.

3. The spun smear. The are two additional types of blood

smear used for specific purposes

1. Buffy coat smear for WBCs < 1.0×109/L and LE cell preparation

2. Thick blood smears for blood parasites

BLOOD SMEAR PREPARATION

Wedge blood smear

Specimen : EDTA blood within 2 to 3 hours & collected to the mark on tube.

Note : May change RBCs morphology such as Spiculated (crenated) cells if :

1. Excessive amount of anticoagulant to specimen

2. Old blood - long standing.

3. Warm environment (room temperature) may hasten changes.

Procedure

Place a drop of blood from mixed sample on a clean glass slide.

Spreader slide using another clean glass slide at 30-40 degree angle.

Control thickness of the smear by changing the angle of spreader slide

Allow the blood film to air-dry completely before staining. (Do not blow to dry. The moisture from your breath will cause RBC artifact.)

STEPS FOR BLOOD FILM PREP

The Thickness of the spread

Notes:1. If the hematocrit is increased, the angle of the

spreader slide should be decreased.2. If the hematocrit is decreased, the angle of

the spreader slide should be increased.

large angle

low HCT

small angle

high HCT

THIN SMEAR

THICK SMEAR

Pressure

Angle

Speed

Size

Characteristics of a Good Smear

1. Thick at one end, thinning out to a smooth rounded feather edge.

2. Should occupy 2/3 of the total slide area.

3. Should not touch any edge of the slide.

4. Should be margin free, except for point of application.

Note: As soon as the drop of blood is placed on the glass slide, the smear should be made without delay. Any delay results in an abnormal distribution of the white blood cells, with many of the large white cells accumulating at the thin edge of the smear.

Size of Blood drop: 2-3 mm Blood from frosted end: 0.25 inch or 1cm End of smear to end of slide: 0.5 inch

(automated spreader)

Common causes of a poor blood smear

1. Drop of blood too large or too small.

2. Spreader slide pushed across the slide in a jerky manner.

3. Failure to keep the entire edge of the spreader slide against the slide while making the smear.

4. Failure to keep the spreader slide at a 30° angle with the slide.

5. Failure to push the spreader slide completely across the slide.

6. Irregular spread with ridges and long tail: Edge of spreader dirty or chipped; dusty slide

7. Holes in film: Slide contaminated with fat or grease

8. Cellular degenerative changes: delay in fixing, inadequate fixing time or methanol contaminated with water.

Examples of unacceptable smears

A: Blood film with jagged tail made from a spreader with a chipped end.

B: Film which is too thick

C: Film which is too long, too wide, uneven thickness and made on a greasy slide.

D: A well-made blood film.

Examples of unacceptable smears

Biologic causes of a poor smear

1. Cold agglutinin - RBCs will clump together. Warm the blood at 37° C for 5 minutes, and then remake the smear.

2. Lipemia - holes will appear in the smear. There is nothing you can do to correct this.

3. Rouleaux - RBC’s will form into stacks resembling coins. There is nothing you can do to correct this.

Notes:1. Although this is the easiest and most popular

methods for producing a blood smear, it does not produce a quality smear.

2. The WBCs are unevenly distributed and RBC distortion is seen at the edges Smaller WBCs such as lymphocytes tend to reside in the middle of the feathered edge.

3. Large cells such as monocytes, immature cells and abnormal cells can be found in the outer limits of this area.

4. Spun smears produce the most uniform distribution of blood cells.

Fixation

To preserve the morphology of the cells, films must be fixed as soon as possible after they have dried.

It is important to prevent contact with water before fixation is complete.

Methyl alcohol (methanol) is the choice, although ethyl alcohol ("absolute alcohol") can be used.

To fix the films, place them in a covered staining jar or tray containing the alcohol for 2-3 minutes.

Staining the film

Romanowsky staining: Romanowsky stains are universally employed for staining

blood films and are generally very satisfactory. There are a number of different combinations of these

dyes, which vary, in their staining characteristics.

1. Wright’s

2. Giemsa: preferred for blood parasites

3. Modified Wright’s-Giemsa

4. Leishman

5. Jenner

6. May-Grunwald

Principle

The main components of a Romanowsky stain are: A cationic or basic dye (methylene blue or its oxidation

products such as azure B), which binds to anionic sites and gives a blue-grey color to nucleic acids (DNA or RNA), nucleoproteins, granules of basophils and weakly to granules of neutrophils

An anionic or acidic dye such as eosin Y or eosin B, which binds to cationic sites on proteins and gives an orange-red color to hemoglobin and eosinophil granules.

pH value of phosphate buffer is very important Blood and BM staining: 6.4-6.8

Malarial parasite staining: 7.2-7.4

Staining procedure

Thin smear are air dried. Flood the smear with stain. Stain for 1-5 min. Experience will indicate the

optimum time. Add an equal amount of buffer solution and mix

the stain by blowing an eddy in the fluid. Leave the mixture on the slide for 10-15 min. Wash off by running water directly to the centre

of the slide to prevent a residue of precipitated stain.

Stand slide on end, and let dry in air.

too acidic suitable too basic

Causes & correction

Too Acid Stain:1. Insufficient staining

2. Prolonged washing time

3. Mounting cover slips before they are dry

4. Too high acidity of the stain

5. Buffer may cause excessive acidophilia

Correction:1. Lengthen staining time

2. Check stain and buffer pH

3. Shorten buffering or wash time

Too Alkaline Stain:

1. thick blood smear

2. prolonged staining

3. insufficient washing

4. alkaline pH of stain components

5. Diluents tend to cause excessive basophilia

 Correction :

1) check pH

2) shorten stain time

3) prolong buffering time

Performing A Manual Differential and Assessing RBC Morphology

White Blood Cells.

1. Check for even distribution and estimate the number present (also, look for any gross abnormalities present on the smear).

2. Perform the differential count.

Principle

Red Blood Cells, Examine for:

1.Size and shape.

2.Relative hemoglobin content.

3.Polychromatophilia.

4. Inclusions.

5.  Rouleaux formation or agglutination

Platelets.

1.Estimate number present.

2. Examine for morphologic abnormalities.

Procedures

Observations Under ×10 (LPO)

1.Check to see if there are good counting areas available free of ragged edges and cell clumps.

2.Check the WBC distribution over the smear.

3.Check that the slide is properly stained.

4.Check for the presence of large platelets, platelet clumps, and fibrin strands.

Observations Under 40x (HPO): WBC Estimates

Using the × 40 high dry with no oil. Choose a portion of the peripheral smear

where there is only slight overlapping of the RBCs.

Count 10 fields, take the total number of white cells and divide by 10.

To do a WBC estimate by taking the average number of white cells and multiplying by 2000.

Observations Under × 100: Platelet Estimates

1. Use the oil immersion lens estimate the number of platelets per field.

2. Look at 5-6 fields and take an average.

3. Multiply the average by 20,000.

4. Note any macroplatelets.

Platelets per oil immersion field (OIF)

1) <8 platelets/OIF = decreased

2) 8 to 20 platelets/OIF = adequate

3) >20 platelets/OIF = increased

PLATELETS

Observing and Recording Nucleated Red Blood Cells (nRBCs)

If 10 or more nucleated RBC's (NRBC) are seen, correct the White Count using this formula:

Corrected WBC Count =

Example : If WBC = 5000 and 10 NRBCs have been counted

Then 5,000× 100/110 = 4545.50

The corrected white count is 4545.50.

Manual Differential Counts

These counts are done in the same area as WBC and platelet estimates with the red cells barely touching.

This takes place under × 100 (oil) using the zigzag method.

Count 100 WBCs including all cell lines from immature to mature.

Reporting results

Absolute number of cells/µl = % of cell type in differential x white cell count

Observing direction:

Observe one field and record the number of WBC according to the different type then turn to another field in the snake-liked direction*avoid repeat or miss some cells

normal peripheral blood smear

Band neutrophil

Segmented neutrophil

1.Neutrophils Neutrophils are so named because they are not well

stained by either eosin, a red acidic stain, or by methylene blue, a basic or alkaline stain.

Neutrophils are also known as "segs", "PMNs" or "polys" (polymorphonuclear).

They are the body's primary defense against bacterial infection.

Increased neutrophils count (neutrophilia) 1. Acute bacterial infection.2. Granulocytic leukemia.

Decreased neutrophil count (neutropenia)3. Typhoid fever4. Brucellosis 5. Viral diseases, including hepatitis, influenza, rubella, and

mumps.

Segmented neutrophil Band neutrophil

Shift to left Increased bands mean acute infection, usually bacterial.

Shift to right Increased hypersegmented neutrophil.

Eosinophil

The most common reasons for an increase in the eosinophil count are

1. Allergic reactions such as hay fever, asthma, or drug hypersensitivity.

2. Parasitic infection

3. Eosinophilic leukemia

Basophil

Basophils The purpose of basophils is not completely understood.

Basophil counts are used to analyze allergic reactions.

An alteration in bone marrow function such as leukemia may cause an increase in basophils.

Lymphocyte

Lymphocytes

Lymphocytes are the primary components of the body's immune system. They are the source of serum immunoglobulins and of cellular immune response.

Two types of lymphocytes:

1. B lymphocyte : Humoral immunity

2. T lymphocyte : Cellular immunity

Lymphocytes increase (lymphocytosis) in: 1.Many viral infections 2.Tuberculosis. 3.Typhoid fever 4.Lymphocytic leukemia. A decreased lymphocyte (lymphopenia) count of less than 500

places a patient at very high risk of infection, particularly viral infections.

Monocyte

Diseases that cause a monocytosis include:•Tuberculosis•Brucellosis•Malaria•Monocytic leukemia

Notes

1. Do not count cells that are disintegrating

• eosinophil with no cytoplasmic membrane and with scattered granules

• Pyknotic cell (nucleus extremely condensed and degenerated, lobes condensed into small, round clumps with no filaments interconnecting).

• smudge cells

• Basket cells

•smudge cells•Basket cells

**WBC COUNTING:

100 cells: Routine

50 cells: pt WBC ct<1x 109/L

200 cells: >10% eosinophils

>2% basophils

>11% monocytes

Lymphocytes > neutrophils (except in

children)

**Absolute # of cells: RELATIVE X WBC/L

tail body head

BLOOD SMEAR PREPARATIONFOR BLOOD PARASITES

Peripheral Smear Preparation for blood parasites

Peripheral smear examination for malarial parasite is the GOLD-STANDARD in confirming the diagnosis of malaria. THICK AND THIN SMEARS prepared from the peripheral blood are used for the purpose.

STEP 1

Hold the third finger of the left hand and clean it with swab dipped in Savlon or dettol

Step 2

Prick the finger with needle or lancet and allow the blood to ooze out.

Step 3

Take a clean glass slide. Take 3 drops of blood 1 cm from the edge of the slide, take another drop of blood one cm from the first drop of blood

Step 4

Take another clean slide with smooth edges and use it as a spreader...

Step 5

...and make thick and thin smears. Allow it to dry

Prepared Smear

Thick Film

The thick smear of correct thickness is the one through which newsprint is barely visible.

It is dried for 30 minutes and not fixed with methanol.

This allows the red blood cells to be hemolyzed and leukocytes and any malaria parasites present will be the only detectable elements.

Thick smears are therefore used to detect infection, and to estimate parasite concentration. (FOR COUNTING)

Thin Film

Air dry the thin smear for 10 minutes. After drying, the thin smear should be

fixed in methanol. This can be done by either dipping the thin smear into methanol for 5 seconds or by dabbing the thin smear with a methanol-soaked cotton ball.

While fixing the thin smear, all care should be taken to avoid exposure of the thick smear to methanol.

FOR SPECIATION.

THIN FILM THICK FILM

ERYTHROCYTE SEDIMENTATION RATE

ERYTHROCYTE SEDIMENTATION RATE (ESR)

A nonspecific screening test indicative of inflammation

There are two methods of determining ESR: Westergren and Wintrobe

Studies have shown that the Wintrobe method was found to be misleading in some important cases

As a result, the Westergren method is most widely used

Erythrocyte sedimentation rate(ESR)

Rate of settling of RBC from the plasma after the addition of anticoagulant.

Importance of ESR

1. Good index for the presence of hidden carcinoma but active diseases.

2. It measures the suspension stability of RBC.

3. It measures the abnormal concentration of fibrinogen and serum globulin.

Roleaux formation

(Packing or piling of RBC)

ERYTHROCYTE SEDIMENTATION RATE (ESR)

Anticoagulated blood is drawn up into a tube of standardized dimensions and left in a vertical position for exactly one hour.

This test measures the distance that RBCs will fall in a vertical tube over a given time period

It is used as an initial screening tool and also as a follow-up test to monitor therapy and progression or remission of disease. The ESR is directly proportional to red cell mass

The ESR is reported in millimeters/hr

ERYTHROCYTE SEDIMENTATION RATE (ESR)

3 STAGES:

1. LAG PHASE: (10 mins) the red cells form a characteristic rouleaux pattern and sedimentation is generally slow.

2. DECANTATION PHASE: (40 mins) sedimentation accelerates.

3. FINAL PACKING PHASE: (10 mins) as red cell aggregates pile up at the base of the tube

ERYTHROCYTE SEDIMENTATION RATE (ESR)

The size of the rouleaux aggregates formed in the Lag Phase is the critical factor affecting the final result of the ESR.

The rouleaux itself appears to be influenced mainly by certain plasma proteins including FIBRINOGEN, IgM AND ALPHA2-MACROGLOBULIN.

Methods

A. Wintrobe and landsberg method Anticoagulant used- Ammonium potassium

oxalate (wintrobe solution/ double oxalate/balanced oxalate/ paul-Heller’s soln.)

Tube – wintrobe tube

- Left side – RED (for ESR: 0-100)

- Right side – WHITE ( for hematocrit: 100-0)

Procedure

1. With a long stem pasteur pipet, fill the wintrobe tube with oxalated blood up to 0 mark.

2. Let the wintrobe tube stand perfectly vertical.

3. Read result after 1 hour. Reading must be done on the left red side of the tube.

Normal values Male - (0-9) mm/hr Female - ( 0-20 )mm/hr Children- (0-13) mm/hr

White 10

Red 0

Red cells

0 at bottom at bottom 10

Layers -Plasma layer-Buffy coat (WBC and platelets)-Packed RBC (hematocrit)

Wintrobe tube

B. Original Westergren method (200mm) – most sensitive and most accurate.

- Anticoagulant used -3.8% sodium citrate

- Tube- westergren tube (through suction method long tube)

200 mm

0

Procedure

1. Fill the tube with the citrated blood

2. Stand the tube vertically and read result at the end of 1 hour.

Normal values

MALE: <50 y/o: 0-15 mm/hr

>50 y/o: 0-20mm/hr

FEMALE: <50 y/o: 0-20 mm/hr

>50 y/o: 0-30mm/hr

CHILDREN: 0-10 mm/hr

Wintrobe Westergren

Bore 3 mm 2.5 mm

Graduation up to 100 mm

up to 200 mm

Anticoagulant Double oxalate

3.8% sodium citrate

Amount of blood

1 ml 2.4 ml

Reading once Twice

Hematocrit

FACTORS AFFECTING ESR

1. Intrinsic Factor

- nos of RBC ( less RBC faster settlement)

- size of RBC ( Bigger the size is faster the settlement)

- viscosity of Plasma ( less viscous fast settlement)

* nos of RBC- inversely

* size of RBC- directly

2. Extrinsic factor Length of tube ( smaller length fast

settlement) Diameter of tube (wider diameter fast

settlement) Position of tube(vertical or slightly fast

settlement Temperature ( high temp. fast settlement) Pipetting ( incorrect pipetting result error) Volume of blood ( less blood faster settle.) Anticoagulant (more anticoagulant slow

settlement)

PATHOLOGICAL CONDITIONS ASSOCIATED WITH ESR

Increased ESR

1. Kidney disease

2. Pregnancy

3. Rheumatic fever

4. Rheumatoid arthritis

5. Anemia

6. Syphilis

7. Systemic lupus erythematosus

8. Thyroid disease

9. Elevated room temperature

10. Ischemic stroke

11. Temporal arteritis

PATHOLOGICAL CONDITIONS ASSOCIATED WITH ESR

Decreased ESR

1. Congestive heart failure

2. Hyperviscosity

3. Decreased fibrinogen levels

4. Polycythemia

5. Sickle cell anemia

< 0.5 mm/hr

RETICULOCYTE COUNT

RETICULOCYTE

The reticulocyte is the cell stage immediately before the mature erythrocyte.

This cell spends 2 to 3 days maturing in the bone marrow before it is released into the peripheral circulation, where it spends an additional day of maturation.

Acidophilic erythroblast (NRBC) expels the nucleus and becomes the reticulocyte

Bone marrow macrophages phagocytize and digest the expelled nucleus.

The size of reticulocyte is about 8 μm

RETICULOCYTEThe reticulocyte count is the most effective measure

of erythropoietic activity.

Reticulocyte counts are a reflection of bone marrow health or injury

Reticulocytes are red cells that are non-nucleated and that contain remnant RNA material, reticulum.

To be counted and evaluated, reticulocytes must be stained with supravital stains, like new methylene blue or brilliant cresyl blue.

RETICULOCYTE

Because the bone marrow has the capacity to expand its production up to 7 times the normal rate, an elevated reticulocyte count or reticulocytosis is the appropriate response in anemic stress

Reticulocytes will be seen in the peripheral smear as polychromatophilic macrocytes (large, bluish cells)

Nucleated red blood cells may also be visualized in the peripheral smear as the bone marrow races to deliver cells prematurely at a rapid rate.

RETICULOCYTE COUNT

Procedure (Supravital Staining)1. Mix 4 drops of New Methylene Blue with 4 drops of patient’s blood.

2. Let the specimen mix for 10 to 15 minutes. Make a wedge smear and let it air dry. Label the smear

3. Allow the smear to completely dry and read under the microscope using x100 oil immersion.

4. Count the number of reticulocytes in 1000 cells (10 fields).

**To be considered a retic, it must contain 2 or more blue-staining particles (reticulum)

RETICULOCYTE

Number of Retic = (# retic per 1000 red cells x 100)/1000Note: 1000 red cells = 10 fields

Example: 35 x100/1000 = 3.5%

OR

Number of retic counted divided by 10

Example; 35/10 = 3.5 %

RETICULOCYTE

RETICULOCYTE

Normal Values

Adults: 0.5% to 1.5%

Infants: 2.0% to 6.0%

RETICULOCYTE

Limitations1. Recent blood transfusion can interfere with accurate reticulocyte results.

2. Mishandling, contamination, or inadequate refrigeration of the sample can interfere and cause inaccurate test results.

3. Red cell inclusions such as Heinz bodies, siderocytes, and Howell-Jolly bodies can be mistaken for reticulocytes. If these are counted as reticulocytes, they will falsely increase the reticulocyte count. Inclusions should be confirmed with Wright’s stain

RETICULOCYTE

Increased Reticulocyte countIncreased reticulocyte counts indicate increased erythropoietic activity usually as the bone marrow compensates in response to anemic stress

Increased count associated with:

1. Rapid blood loss

2. High elevation

3. Hemolytic anemias

4. Medications such as levodopa, malarial medications, corticotrophin, and fever-reducing medications

5. Pregnancy

RETICULOCYTE

Decreased Reticulocyte count (<0.5%)

Low reticulocyte counts indicate decreased erythropoietic activity

Decreased count associated with:

1. Aplastic anemia (where the production of either white or red cells or both is seriously impaired)

2. Exposure to radiation or radiation therapy

3. Chronic infection4. Medications such as chloramphenicol, methotrexate and other chemotherapy medications5. Untreated pernicious anemia/megaloblastic anemia

RETICULOCYTE

Calculation of corrected reticulocyte

The reticulocyte count is most often expressed as a percentage of total red cells.

In states of anemia, the reticulocyte percentage is not a true reflection of reticulocyte production.

A correction factor must be used so as not to overestimate marrow production

Because each reticulocyte is released into whole blood containing few RBCs and a low hematocrit (Hct), the percentage of reticulocyte will be increased.

RETICULOCYTE

Calculation of corrected reticulocyteThe corrected reticulocyte count may be calculated

by the following formula:

Corrected reticulocyte count = (Retic % x patient’s Hct %) divided by Average normal Hct

Note: Average normal Hct is 45%. 

Example: Uncorrected retic % = 5.0%Patient Hct = 35.0% (male)Average normal hct for male = 45%

Corrected retic % = (5.0% x 35%) / 45 = 3.89%

Corrected retic % = 175/45 = 3.89 %

Calculation of reticulocyte production index (RPI)

general indicator of the rate of erythrocyte production increase above normal in anemias.

indicated BM response to anemia

RPI= CRC ÷ maturation time of retics in the blood NV: 1 (Hct: 45%)

 

**maturation time of retics in blood1 day= Hct: 45 ± 5%1.5 days= Hct: 35 ± 5%2 days= Hct: 25 ± 5%2.5 days= Hct: 15 ± 5%

RPI >3 RPI <2Adequate response of BM to anemia

Inadequate response of BM to anemia

- Chronic hemolysis - Aplastic anemia

- Recent hemorrhage - Ineffective erythropoiesis (megaloblastic anemia)

- Response to therapy  

EOSINOPHIL COUNT

EOSINOPHIL COUNTNV: 50-350 x 109/L

Allergic reactions

Hyperadrenalism (Cushing’s disease)

Parasitic infections

Shock

Brucellosis Ff. administration of ACTH

Certain leukemias

 

2 METHODS:

1. INDIRECT: WBC x %eos

2. DIRECT: most widely used

DILUTING FLUID:

- Phloxine/Eosin/Neutral red iodide: stain EOS red.

- Propylene glycol: lyses RCs

- Na2CO3: lyse WBCs except eos.

- Heparin: prevents clumping

 

**Fuch’s Rosenthal counting chamber: recommended

THORN’S TEST

assesses adrenocortical function

EO Ct #1: FASTING

EO Ct #2: 4 hours after administration of ACTH (normally, )

NORMAL: EO Ct #1 > EO Ct #2 lower by 50%

HYPOADRENALISM: EO Ct #1 = EO Ct #2

 

SICKLE CELL TESTS

A. SODIUM METABISULFITE METHOD

*WB + Sodium metabisulfite (strong RA) SICKLING (maximal at 37

 

@HPO after 1 hour to 24 hours at room temperature.

POSITIVE: presence of holly-leaf form (sickle cell)

B. SOLUBILITY TEST

- uses 12x 75mm test tubes

- tubes are approximately 1 inch in front of the lined reader scale.

 

*RBCs + saponin (lyses RBCs) Hgb S forms liquid crystals causing turbidity.

POSITIVE: turbid, lines are not visible

NEGATIVE: solution remains clear

FALSE (+) FALSE (-)- Inc

immunoglobulins- Use of 10x75 mm

test tube- Lipemia - <7 g/dl hgb- Inc WBCs  

END