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. II. III. IV. Introduction Specimen Collection Material for Cell Counting 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

Collect the following blood

Wipe off the first drop of blood

VENIPUNCTURE
Clean site using alcohol Tie tourniquet 2in. above the site

Open close the fist of the patient

Check the syringe:

Plunger, needle (hub)

Draw blood

Transfer blood to the intended tube

Materials for Cell Counting

Hemocytometer / Counting Chamber
Fuchs Rosenthal Speirs Levy Neubauer Improved Neubauer

Materials for Cell Counting

Thoma Pipette
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

 Hematology

Different Laboratory Examinations

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

Diluting the blood

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

Charging the counting chamber

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

Counting the cells

LPO, count on the 4 Secondary squares Add all cells

Compute

Results
Group 1 2 3 4 5 Result 6.93x 106 cells/ mL 5.37x 106 cells/ mL 5.16x 106 cells/ mL 4.68x 106 cells/ mL 5.31x 106 cells/ mL Interpretation Increased Normal Normal Normal Normal

Results
Group Result Interpretation

6 7
8 9 10

5.34 x 106 cells/mL 5.18 x 106 cells/mL

5.20 x 106 cells/mL 5.62 x 106 cells/mL 5.30 x 106 cells/mL

Normal Normal
Normal Normal 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 Hydantoins Chloramphenicol Quinidine

THREE MAIN CAUSES OF ANEMIA

1. BLOOD LOSS Most common cause; IDA E.g. heavy menstrual flow, GI/UT bleeding, surgery, trauma, cancer

THREE MAIN CAUSES OF ANEMIA

2. LACK OF RBC PRODUCTION Acquired poor diet, abnormal hormone levels, chronic dse, pregnancy Inherited aplastic anemia*

THREE MAIN CAUSES OF 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

OTHER CAUSES OF ANEMIA

2. DISEASES & DISEASE TREATMENTS Chronic diseases (kidney disease and cancer) Cancer treatments; HIV/AIDS medicines

OTHER CAUSES OF ANEMIA

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

Diluting the blood

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

Charging the counting chamber

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

Counting the cells

HPO, count on the 5 squares of the 25 small squares

Add all cells

Compute

Results
Group 1 2 3 4 5 Result 6.35 x 103 cells/ mL 6.65 x 103 cells/ mL 8.30 x 103 cells/ mL 5.75 x 103 cells/ mL 7.50 x 103 cells/ mL Interpretation Normal Normal Normal Normal Normal

Results
Group 6 7 8 9 10 Result 7.0 x 103 cells/mL 8.1 x 103 cells/mL 6.8 x 103 cells/mL 7.2 x 103 cells/mL 7.3 x 103 cells/mL Interpretation Normal Normal Normal Normal Normal

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 Adams Method micro method

1% hematocrit = 0.34 gm% hemoglobin = 107, 000 RBC/cumm

Filled a capillet with blood

Seal one end

(two thirds filled)

Measure using hematocrit reader

Centrifuge for 5 minutes

Results
Group 1 2 3 4 5 Result 58% 57% 40% 47% 45% Interpretation Increased Increased Normal Normal Normal

Results
Group 6 7 8 9 10 Result 48% 50% 46% 43% 46% Interpretation Normal Normal Normal Normal Normal

HEMOGLOBIN DETERMINATION
Cyanmethemoglobin
Use of Drabkins Reagent Ferriccyanide in the Drabkins 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 50-70% 2-6%

Segmented Neutrophil Neutrophilic Band Lymphocytes

Nucleus: broken into segments Cytoplasm: small, pinkish granules Younger neutrophil Nucleus: C,S,U of horse-shoe shaped Nucleus: compact, round Cytoplasm: light blue, scanty Largest, vacuoles are sometimes present Nucleus: spongy, sprawling w/ brain-like convulutions Cytoplasm;:light gray
Nucleus: bilobed Cytoplasm: large/course reddish/ pinkish/ orange granules

20-40%

Monocyte

2-8%

Eosinophil

1-4%

Basophil

Least numerous Nucleus: indistinct, obscured by granules Cytoplasm: large purplish-black/ dark-blue granules

0-1%

Results
WBC Neutrophil Lymphocyte Monocyte Eosinophil 1 56 32 9 3 2 61 27 8 4 3 67 23 6 4 4 60 35 3 2 5 55 37 5 3

Basophil

Results
WBC Neutrophil Lymphocyte Monocyte Eosinophil 6 60 35 3 2 7 64 33 2 1 8 59 31 7 3 9 57 39 4 0 10 61 30 8 1

Basophil

WBC Disorders

Neutrophilia

An 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

Neutropenia

A 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 disorders Diseases that increase neutrophil destruction include: Splenomegaly with hypersplenism Infection Immune destruction

WBC Disorders

Lymphocytosis

An 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

Lymphopenia

A 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

Monocytosis

An 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

Monocytopenia

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

WBC Disorders

Eosinophilia

An 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

Eosinopenia

An 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

Dukes Method
Skin puncture at the finger Uses filter paper for blotting off blood

Ivys 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
Group 1 2 3 4 5 Result 1 min. 1min. 30sec. 1min. 30 sec. 1min. 30sec. 1min. 30 sec. Interpretation Normal Normal Normal Normal Normal

Results
Group 6 7 8 9 10 Result 2min. 30sec. 2min. 3 min. 2min. 30sec. 3min. Interpretation Normal Normal Normal Normal Normal

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

BRIEF EXPLANATION : In order for blood to clot, the enzyme thrombin must be generated from the plasma precursor prothrombin.
Thrombin then converts soluble 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
Group 1 2 3 4 5 Result 1min. 30sec. 3min. 30sec. 6min. 4min 30 sec. 3min. 30 sec. Interpretation Decreased Normal Normal Normal Normal

Results
Group 6 7 8 9 10 Result 6min. 30sec. 5min. 5min. 30sec. 7min. 3min. Interpretation Normal Normal Normal Normal 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 aspartate aminotransferase 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 DISEASE is a group of hereditary genetic disorders that impair the body's ability to control blood clotting or coagulation, which is used to stop bleeding when a blood vessel is broken. VON WILLEBRAND DISEASE is the most common hereditary coagulation abnormality described in humans, although it can also be acquired as a result of other medical conditions. It arises from a qualitative or quantitative deficiency of von Willebrand factor, a multimeric protein that is required for platelet adhesion to exposed collage of blood vessels.

ABNORMALITIES/PATHOLOGIC CHANGES EVIDENT IN:

SEVERE ANEMIA In patients with anemia, there is a change in the distribution of platelets and a decreased interaction of the platelets with the vascular endothelium resulting in a prolonged BT. Correction of the anemia will improve the BT.
THROMBOCYTOPENIA One common definition of thrombocytopenia is a platelet count below 50,000 per microlitre Inspection 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.

ABNORMALITIES/PATHOLOGIC CHANGES EVIDENT IN:

CLOTTING FACTORS DEFICIENCY Factor II deficiency (Prothrombin), Factor V deficiency (Proaccelerin), Factor XI deficiency (plasma thromboplastin antecedent), Factor XII (hageman factor) CONGENITAL AFIBRINOGEMIA is a rare inherited blood disorder in which the blood does not clot normally due to a lack of or a malfunction involving 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

Dislodge and check for agglutination

Centrifuge

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

Dislodge and check for agglutination

Centrifuge

Rh Typing
Slide Method
3rd drop of blood
Add Anti-D Mix, rotate and check for agglutination

Tube Method
5%red cell suspension Add Anti-D; Centrifuge Dislodge and check for agglutination

Forward Typing
Blood Group A B AB O Anti-A + + Anti-B + + Anti-AB + + + -

Reverse Typing
Blood Group A B AB O A-Cells + + B-Cells + + AB-Cells + + +

Results:
Group Result Anti-A, Anti-B, & Anti-D: Positive 1
2
Anti-A & Anti-B: Negative Anti-D: Positive Anti-A: Positive Anti-B: Negative Anti-D: Positive Anti-A: Positive Anti-B: Negative Anti-D: Positive Anti-A & Anti-B: Negative Anti-D: Positive

Interpretation AB+
O+

3
4

A+
A+

O+

Results:
Group 6 7 8 9 Result
Anti-A & Anti-B: Negative Anti-D: Positive Anti-A: Negative Anti-B & Anti-D: Positive Anti-A: Negative Anti-B & Anti-D: Positive Anti-A & Anti-B: Negative Anti-D: Positive Anti-A & Anti-B: Negative Anti-D: Positive

Interpretation
O+
B+ B+ O+ O+

10

Cross-matching
Major Crossmatch
Patients Serum and Donors Red Cell (PSDR)

Minor Crossmatch
Donors Serum and Patients Red Cell (DSPR)

Saline Phase
Major Crossmatch
Mix patients serum and donors red cell centrifuge; dislodge Check for agglutination or hemolysis Negative: continue to THERMO PHASE

Minor Crossmatch
Mix patients red cell and donors serum

centrifuge; dislodge

Check for agglutination or hemolysis Negative: continue to THERMO PHASE

Thermo Phase

Incubate tubes

Centrifuge; dislodge

Check for agglutination or 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

No agglutination, add check cells

Check for agglutination or hemolysis

Centrifuge and dislodge

Slide Method

Mix patient serum and donors whole blood in the slide

Mix, rotate and check for agglutination

With agglutination: Incompatible No agglutination: compatible

Results
Whole Blood Serum Result Interpretation

Agglutination

Incompatible

3
5 7

4
6 8

Agglutination
No agglutination Agglutination

Incompatible
Compatible Incompatible

10

No agglutination

Compatible

Significance of Cross-matching
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

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

Transfusion Reactions
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 IgE mast cell activation Itchiness/Urticaria