red cell and white cell counting, blood

Red Cell and White Cell Counting, Blood Typing, Cross-matching, and

Osmotic Fragility Test

AUF- SOM 2016

Section A

Reporters

Group 3

• Leader:

• Members:

Group 8

• Leader:

Angelyka Francisco

• Members:

Nina Gamboa

Jovel Gangcuangco

John Glorie

Bryan Gozum

Beejay Garcia

Outline

I. Introduction

II. Specimen Collection

III. Material for Cell Counting

IV. Different Laboratory Examinations and Results

Introduction

• Blood

– Composed of:

• Plasma

• Blood Cells

Introduction

• WBC Count– Number of WBC in a liter of whole blood– NV: 4-113 x 103 cells/ mL

• RBC Count:– Rarely performed due to inaccuracy of the count and

questionable necessity– NV: Male: 4.5-6.53 x 106 cells/ mL

Female: 3.99-5.63 x 106 cells/ mL

• Platelet Count:– Used in a phase-contrast microscope– NV: 200-5,003 x 103 cells/ L

Introduction

• Hematocrit– Measurement of the packed RBC

– NV: Male: 40-50%

Female: 37-48%

• Hemoglobin– Measurement of iron content of RBC in relation to

its oxygen binding capacity

– NV: Male: 14-17 g/dL

Female: 12- 16 g/dL

Introduction

• Differential WBC count– Process of differentiating and having a rough estimate

of the percentage of the different WBC that circulate in the body

• Bleeding Time– Ability of small vessels to control bleeding

– Affected by aspirin

• Clotting Time– Period required for free flowing blood to clot or

solidify after it has been removed from the body

Specimen Collection

• Capillary or Skin Puncture

• Venipuncture

• Arterial Puncture

Capillary/Skin Puncture

Massage intended site

Clean site with alcohol

Using a lancet, one quick jab

Wipe off the first drop of

blood

Collect the following

blood

Clean site using alcohol

Tie tourniquet 2in. above the site

Check the syringe:

Plunger, needle (hub)

Open close the fist of the patient

Draw bloodTransfer blood to the

intended tube

VENIPUNCTURE

Materials for Cell Counting

• Hemocytometer / Counting Chamber

– Fuch’s Rosenthal

– Speir’s Levy

– Neubauer

– Improved Neubauer

Materials for Cell Counting

WBC Pipette

• 11 mark

• White bead

• Bigger bore

• 10 Units volume

• Produce 1:10 and 1:20 dilution

RBC Pipette

• 101 mark

• Red bead

• Smaller bore

• 100 Units volume

• Produce 1:100 and 1:200 dilution

Thoma Pipette

Different Laboratory Examinations• Hematology

– Red Cell Count– White Cell Count– Platelet Count– Hematocrit Determination– Hemoglobin Determination– Differential WBC count– Bleeding Time– Clotting Time

• Blood Banking– Blood Typing– Cross-matching

RED CELL COUNT

• Use isotonic solution: weak solution of NSS

• Uses WBC Thoma Pipette

• 1:20 dilution

Compute

Counting the cells

LPO, count on the 4 Secondary squares Add all cells

Charging the counting chamber

Shake pipette (2-3 mins.) Discard first 4 drops. Fill up the counting chamber

Diluting the blood

Suck blood up to 0.5 mark Suck diluting fluid from 0.5 mark up to 11 mark

Results

Group Result Interpretation

1 6.93x 106 cells/ mL Increased

2 5.37x 106 cells/ mL Normal




Results


6 5.34 x 106 cells/mL Normal





FACTORS THAT ALTER NORMAL RBC COUNT

HIGHER-THAN-NORMAL

• Cigarette smoking

• Congenital heart disease

• Cor pulmonale

• Dehydration (severe diarrhea)

• Kidney tumor (renal cell carcinoma)

• Low blood O2 levels (hypoxia)

HIGHER-THAN-NORMAL

• Pulmonary fibrosis

• Polycythemia vera

• Drugs

–Gentamicin

–Methyldopa

LOWER-THAN-NORMAL

• Anemia

• Bone marrow failure

• Erythropoietin deficiency

• Hemolysis (RBC destruction)

• Hemorrhage

• Leukemia

LOWER-THAN-NORMAL

• Malnutrition

• Multiple myeloma

• Nutritional Deficiencies:

• Iron• Copper• Folate

• Vitamin B12• Vitamin B6

LOWER-THAN-NORMAL

• Overhydration

• Pregnancy

• Drugs

• Chemo. drugs• Chloramphenicol

• Hydantoins• Quinidine

THREE MAIN CAUSES OF ANEMIA

1. BLOOD LOSS

• Most common cause; IDA

• E.g. heavy menstrual flow, GI/UTbleeding, surgery, trauma,cancer


2. LACK OF RBC PRODUCTION

• Acquired – poor diet, abnormalhormone levels, chronic dse,pregnancy

• Inherited – aplastic anemia*


3. HIGH RATES OF RBC DESTRUCTION

• Acquired – enlarged/diseased spleen

• Inherited – sickle cell anemia, thalassemia, lack of certain enzymes, hemolytic anemia*

OTHER CAUSES OF ANEMIA

1. DIET

• Iron, folic acid (folate), Vit. B12, Vit. C, riboflavin, copper

• Problems in nutrient absorption


2. DISEASES & DISEASE TREATMENTS

• Chronic diseases (kidney disease and cancer)

• Cancer treatments; HIV/AIDS medicines


2. PREGNANCY

• Low iron and folic acid levels

• Hemodilution

3. Aplastic Anemia

WHITE CELL COUNT

• Use hypotonic solution: weak solution of acetic acid

• Uses RBC Thoma Pipette

• 1:200 dilution

Compute

Counting the cellsHPO, count on the 5 squares of the 25 small

squaresAdd all cells

Charging the counting chamber

Shake pipette (2-3 mins.) Discard first 4 drops. Fill up the counting chamber

Diluting the blood

Suck blood up to 0.5 markSuck diluting fluid from 0.5 mark up to 101

mark

Results


1 6.35 x 103 cells/ mL Normal





Results







PLATELET COUNT

• Uses hypotonic or isotonic solution

• Uses the Rees-Ecker diluting fluid

• As reference method : uses phase-contrast microscope in counting

• 1:200 dilution

HEMATOCRIT DETERMINATION

• Wintobe Method – macro method

• Adam’s Method – micro method

1% hematocrit = 0.34 gm% hemoglobin

= 107, 000 RBC/cumm

Filled a capilletwith blood

(two thirds filled)

Seal one end

Centrifuge for 5 minutes

Measure using hematocrit

reader

Results


1 58% Increased

2 57% Increased

3 40% Normal

4 47% Normal

5 45% Normal

Results


6 48% Normal

7 50% Normal

8 46% Normal

9 43% Normal

10 46% Normal

HEMOGLOBIN DETERMINATION

• Cyanmethemoglobin

– Use of Drabkin’s Reagent

– Ferriccyanide in the Drabkin’s Reagent converts the iron in the Hemoglobin molecule from the ferrous to ferric state forming methemoglobin/ hemiglobin/ ferrihemoglobin, its product combines with potassium cyanide to produce cyanmethemoglobin

Rule of Three

• The value of the Hb should be three times the RBC count, and the Hct shouldd be three times the value of the Hb plus or minus 3.

• RBC x 3 = Hb

• Hb x 3 = Hct ± 0.03

DIFFERENTIAL WBC COUNT

• Wright Staining:

– Solution 1: Methanol (fixative)

– Solution 2: Eosin (acidic dye)

– Solution 3: Methylene Blue (basic dye)

– Buffer Solution pH 7.2

WBC Description Normal Value

Segmented Neutrophil

Nucleus: broken into segmentsCytoplasm: small, pinkish granules

50-70%

NeutrophilicBand

Younger neutrophilNucleus: C,S,U of horse-shoe shaped

2-6%

LymphocytesNucleus: compact, roundCytoplasm: light blue, scanty

20-40%

Monocyte

Largest, vacuoles are sometimes presentNucleus: spongy, sprawling w/ brain-like convulutionsCytoplasm;:light gray

2-8%

EosinophilNucleus: bilobedCytoplasm: large/course reddish/ pinkish/ orange granules

1-4%

Basophil

Least numerousNucleus: indistinct, obscured by granulesCytoplasm: large purplish-black/ dark-blue granules

0-1%

Results

WBC 1 2 3 4 5

Neutrophil 56 61 67 60 55

Lymphocyte 32 27 23 35 37

Monocyte 9 8 6 3 5

Eosinophil 3 4 4 2 3

Basophil 0 0 0 0 0

Results

WBC 6 7 8 9 10

Neutrophil 60 64 59 57 61

Lymphocyte 35 33 31 39 30

Monocyte 3 2 7 4 8

Eosinophil 2 1 3 0 1

Basophil 0 0 0 0 0

WBC Disorders

NeutrophiliaAn increase in the absolute neutrophil count, it can be increased transiently with

stress and exercise by a shift of neutrophils from the marginating pool to the circulating pool. Pathologic processes that result in neutrophilia include:

Infection

Toxins: metabolic (uremia), drugs, chemicals

Tissue destruction or necrosis: infarction, burns, neoplasia, etc

Hemorrhage, especially into a body cavity

Rapid hemolysis

Hematologic disorders: leukemias, myeloproliferative disorders

WBC Disorders NeutropeniaA decrease in the absolute neutrophil count. Pathologic processes that result in

neutropenia include processes that decrease production or increase destruction. Diseases that decrease neutrophil production include:

Aplastic anemia

Toxins that damage marrow

Collagen vascular diseases (such as SLE)

Myelphthisic marrow processes such as marrow infiltration by infections or metastatic carcinomas

Hematologic malignancies such as leukemias

Myeloproliferative disorders

Radiation therapy

Chemotherapy Congenital disordersDiseases that increase neutrophil destruction include: Splenomegaly with hypersplenism Infection Immune destruction

WBC Disorders

LymphocytosisAn increase in the number of circulating lymphocytes may normally be observed in

infants and young children. Pathologic processes with lymphocytosis may include:

Acute infections, including pertussis, typhoid, and paratyphoid

Infectious mononucleosis, with "atypical" lymphocytosis

Viral infections, including measles, mumps, adenovirus, enterovirus, and Coxsackie virus

Toxoplasmosis

HTLV I

WBC Disorders

LymphopeniaA decrease in the number of circulating monocytes may be seen with

Immunodeficiency syndromes, including congenital (DiGeorge syndrome, etc) and acquired (AIDS) conditions

Corticosteroid therapy

Neoplasia, including Hodgkin's disease, non-Hodgkin's lymphomas, and advanced carcinomas

Radiation therapy

Chemotherapy

WBC Disorders

MonocytosisAn increase in the number of circulating monocytes may be seen with

Infections: such as brucellosis, tuberculosis and rickettsia

Myeloproliferative disorders

Hodgkin's disease

Gastrointestinal disorders, including inflammatory bowel diseases and sprue

MonocytopeniaA decrease in the number of circulating monocytes may be seen with: Early corticosteroid therapy Hairy cell leukemia

WBC Disorders

EosinophiliaAn absolute increase in the number of circulating eosinophils may occur with:

Allergic drug reactions

Parasitic infestations, especially those with tissue invasion

Extrinsic asthma

Hay fever

Extrinsic allergic alveolitis ("farmer's lung")

Chronic infections

Hematologic malignancies: CML, Hodgkin's disease

EosinopeniaAn absolute decrease in the number of circulating eosinophils may occur with: Acute stress reactions with increased glucocorticoid and epinephrine secretion Acute inflammation Cushing's syndrome with corticosteroid therapy

WBC Disorders

Basophilia and Basopenia An absolute increase in the number of circulating basophils may occur with

myeloproliferative disorders and with some allergic reactions.

An absolute decrease in the number of circulating basophils may occur with the same conditions that lead to eosinopenia.

Bleeding Time

• Bleeding time is a medical test done on someone to assess their platelet function for certain disease

• The test is dependent upon an adequate number of functionally active platelets that can adhere to the endothelium to form aggregates.

Bleeding Time - Methods

• Duke’s Method– Skin puncture at the finger– Uses filter paper for blotting off blood

• Ivy’s Method– Uses sphygmomanometer– Skin puncture at the arm– Uses filter paper for blotting off blood– NV: 1-7minutes

• Copley-Lalitch Method– Skin puncture at the finger– Immersed the punctured finger at a physiologic saline

solution warmed at 37oC

Apply sphygmomanometer; inflate at 40 mmHg

Clean site(free from veins)

Make 2 successive punctures using a lancet

Blot filter paper every 30 seconds until bleeding stops

Normal Value

The reference range for this test is between 2-9 minutes. In cases in which the BT exceeds 20 minutes it is usual to stop at 20 minutes and report the BT as >20minutes.

Results


1 1 min. Normal

2 1min. 30sec. Normal

3 1min. 30 sec. Normal



Results



7 2min. Normal

8 3 min. Normal


10 3min. Normal

CLOTTING TIME

• It is the time required for the blood to clot in vitro at a temperature of 37°C.

BRIEF EXPLANATION :

•In order for blood to clot, the enzyme thrombin must be generated from the plasma precursor prothrombin.

•Thrombin then convertssoluble fibrinogen into insoluble fibrin.

CLOTTING TIME

• The time taken for blood to clot mainly reflects the time required for the generation of thrombin

• If the plasma concentration of prothrombin, or some of the other factors, is low (or if the factor is absent, or functionally inactive), clotting time will be prolonged.

• The expected range for clotting time/normal value is : 4-10 mins.

CLOTTING TIME - Procedures

• Drop or Slide Method

– Blood is directed to the slide after puncture

– NV: 2-4 minutes

• Capillary Tube or Dale and Laidlaws Method

– Uses non-heparinized capillary tube

– Letting it stay in horizontal position for 2mins. Then break 1cm of the tube every succeeding 30secs.

Results


1 1min. 30sec. Decreased


3 6min. Normal

4 4min 30 sec. Normal


Results



7 5min. Normal


9 7min. Normal

10 3min. Normal

Clinical Significance

Find a cause for abnormal bleeding or bruising.

Check if blood-thinning medicine, such as warfarin (Coumadin), is working.

Check for low levels of blood clotting factors. The lack of some clotting factors can cause bleeding disorders such as hemophilia, which is passed in families (inherited).

Clinical Significance

Check for a low level of vitamin K. Vitamin K is needed to make prothrombin and other clotting factors.

Check how well the liver is working. Clotting levels are checked along with other liver tests, such as aspartateaminotransferase and alanine aminotransferase.

Check to see if the body is using up its clotting factors so quickly that the blood can't clot and bleeding does not stop. This may mean the person has disseminated intravascular coagulation (DIC).

ABNORMALITIES/PATHOLOGIC CHANGES EVIDENT IN:

HEMOPHILIA & CHRISTMAS DISEASEis a group of hereditary genetic disorders that impair thebody's ability to control blood clotting or coagulation,which is used to stop bleeding when a blood vessel isbroken.

VON WILLEBRAND DISEASEis the most common hereditary coagulation abnormalitydescribed in humans, although it can also be acquired asa result of other medical conditions. It arises from aqualitative or quantitative deficiency of von Willebrandfactor, a multimeric protein that is required for plateletadhesion to exposed collage of blood vessels.


SEVERE ANEMIA

In patients with anemia, there is a change in thedistribution of platelets and a decreased interaction ofthe platelets with the vascular endothelium resulting in aprolonged BT. Correction of the anemia will improve theBT.

THROMBOCYTOPENIA

One common definition of thrombocytopenia is a platelet count below 50,000 per microlitreInspection typically reveals evidence of slow, continuous bleeding from any injuries or wounds.

DISSEMINATED INTRAVASCULAR COAGULATION

• In DIC, the processes of coagulation and fibrinolysis are dysregulated, and the result is widespread clotting with resultant bleeding. Regardless of the triggering event of DIC, once initiated, the pathophysiology of DIC is similar in all conditions. One critical mediator of DIC is the release of a transmembrane glycoprotein called tissue factor (TF). TF is present on the surface of many cell types (including endothelial cells, macrophages, and monocytes) and is not normally in contact with the general circulation, but is exposed to the circulation after vascular damage.


CLOTTING FACTORS DEFICIENCY

Factor II deficiency (Prothrombin), Factor V deficiency(Proaccelerin), Factor XI deficiency (plasma thromboplastinantecedent), Factor XII (hageman factor)

CONGENITAL AFIBRINOGEMIA

is a rare inherited blood disorder in which the blood does notclot normally due to a lack of or a malfunctioninvolving fibrinogen, a protein necessary for coagulation.

OTHERS:

leishmaniasis, SLE, smallpox & toxic effects of venom

BLOOD TYPING

• Forward Typing– Uses commercially-prepared antisera of known

specificity• Slide Method – uses whole blood• Tube Method – uses 2-5% RBC suspension

• Reverse Typing– Detection of unknown antibodies present in the

patient serum using red cells of known antigenic specificity

• Rh Typing– Detection of D antigen on the red cells– Rh positive or Rh Negative

Forward Typing: Slide Method

Collect blood through skin puncture

Make 3 drops of blood in a slide

Add the anti-sera. Mixed then rotate.

Agglutination: Positive

No Agglutination: Negative

Forward Typing: Tube Method

Prepare 2-5% RC suspension of the Sample

Add anti-sera to the tubes

Add the cell suspension

Centrifuge Dislodge and

check for agglutination

Reverse Typing

Prepare 2-5% RC suspension of the

A, B, AB and O Cells

Add cell suspension into different tubes

Add serum of the sample

Centrifuge Dislodge and check

for agglutination

Rh Typing

• Slide Method

• Tube Method

3rd drop of blood

Add Anti-DMix, rotate

and check for agglutination

5%red cell suspension

Add Anti-D; Centrifuge

Dislodge and check for agglutination

Forward TypingBlood Group Anti-A Anti-B Anti-AB

A + - +

B - + +

AB + + +

O - - -

Reverse Typing

Blood Group A-Cells B-Cells AB-Cells

A - + +

B + - +

AB - - -

O + + +

Results:Group Result Interpretation

1 Anti-A, Anti-B, & Anti-D: Positive AB+

2Anti-A & Anti-B: Negative

Anti-D: PositiveO+

3Anti-A: PositiveAnti-B: NegativeAnti-D: Positive

A+

4Anti-A: PositiveAnti-B: NegativeAnti-D: Positive

A+

5Anti-A & Anti-B: Negative

Anti-D: PositiveO+

Results:


6 Anti-A & Anti-B: NegativeAnti-D: Positive

O+

7 Anti-A: NegativeAnti-B & Anti-D: Positive

B+

8 Anti-A: NegativeAnti-B & Anti-D: Positive

B+

9 Anti-A & Anti-B: Negative Anti-D: Positive

O+

10 Anti-A & Anti-B: NegativeAnti-D: Positive

O+

Cross-matching

• Major Crossmatch

– Patient’s Serum and Donor’s Red Cell (PSDR)

• Minor Crossmatch

– Donor’s Serum and Patient’s Red Cell (DSPR)

Saline Phase• Major Crossmatch

Mix patient’s serum and donor’s red cell

centrifuge; dislodge

Check for agglutination or

hemolysis

Negative: continue to THERMO PHASE

• Minor Crossmatch

Mix patient’s red cell and donor’s

serumcentrifuge; dislodge


hemolysis

Negative: continue to THERMO PHASE

Thermo Phase

Incubate tubes Centrifuge; dislodge


hemolysis

Negative: continue to AHG Phase

Antihuman Globulin Phase

Wash cells with NSS (3x)

Decant NSS completely on

the last washing

Add the antihuman

globulin sera

Centrifuge and dislodge


hemolysis

No agglutination,

add check cells

Slide Method

Mix patient serum and donor’s whole blood in the slide

Mix, rotate and check for

agglutination

With agglutination: Incompatible

No agglutination: compatible

Results

Whole Blood Serum Result Interpretation

1 2 Agglutination Incompatible


5 6 No agglutination Compatible


9 10 No agglutination Compatible

• Series of procedures performed prior to a blood transfusion

• Determine if the donor's blood is compatible with the blood of an intended recipient

• Purposes:- Ensure maximum benefit to recipient- Avoid possible transfusion reactions

Significance of Cross-matching

Most common errors in Cross-matching:

• Clerical/Technical Errors• mislabeling• improper washing of RBCs• misinterpretation of reactions

Transfusion Reactions

• Hemolytic transfusion reaction• Destruction of donor RBCs by preformed

recipient antibodies (intravascular hemolysis)• Most dangerous complication high mortality• Symptoms:

• hemoglobinuria, fever, chills, chest pain, backache, increased heart rate, shortness of breath, rapid drop in BP, etc.

• Immediate HTR• Within 48 hours

• Delayed HTR• After 5-7 days

• Febrile Nonhemolytic Transfusion Reactions• due to acquired antibodies to donor leukocyte

antigens or pyrogenic cytokines elaborated by leukocytes present in the blood components

• hyperthermia during or after transfusion

• Allergic/Anaphylactic Transfusion Reaction• within minutes of transfusion• most frequent (1-2% of transfusions)• allergen (protein in transfused plasma) binds to

recipient IgEmast cell activation• Itchiness/Urticaria

Transfusion Reactions

red cell and white cell counting, blood

Documents

counting chambershake

anti

absolute neutrophil

disseminated

forward typing

filter paper

myeloproliferative

clotting time