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In alpha thalassemia major, anemia is actually fatal. Red blood cell (RBC) count is increased while hemoglobin is severely decreased. Both the MCV and MCHC are decreased. Red cell distribution width (RDW) is increased. RBC morphology shows slight hypochromic microcytosis with codocytes, schizocytes, nucleated RBCs. Reticulocytes are increased.Hemoglobin electrophoresis demonstrates abnormal pattern on cord blood: Hb A - absentHb Bart's - 80-90%Hb Portland - 0-20%Note: Bone marrow demonstrates marked erythroid hyperplasia.

A 42-year-old male received 6 units of RBCs during open heart surgery 6 months ago. His antibody screen was negative at that time. He has returned for a follow up surgery and his antibody screen is now positive with both screen cells at the AHG phase.Reactions are occurring at AHG phase, which indicates a possible clinically significant antibody, Jka showing dosage. Refer to Case Study 1 panel below to see reactions of antibody panel.IS = Immediate Spin; AHG = Antihuman Globulin Phase; CC = Check Cells; AC = Auto Control; ND= Not doneCase study 1 conclusion:Patient's previous transfusion 6 months ago exposed him to the Jka antigen, causing the formation of this antibody, which is known for showing dosage.

Monospecific anti-human globulin (IgG) enables sensitized red cells to cross-link so that agglutination is visible.Enhancement media are sometimes used to further promote agglutination and reduce incubation time. Low ionic strength saline (LISS) is the most common enhancement media. LISS reduces the ionic strength in the testing sample and causes reduction of the zeta potential. It increases antibody uptake and decreases incubation time. Polyethylene glycol (PEG): brings red blood cells (RBCs) closer together and concentrates antibodies by removing water molecules from the testing sample. It is the most sensitive of the enhancement media; strengthening almost all clinically significant antibodies. However, it will also enhance some clinically insignificant antibodies as well. Centrifugation should be avoided when PEG is used. PEG can cause aggregates to form if the sample (red cell - serum mixture) with PEG added is centrifuged. Reaction readings should only be done at the AHG phase. 22% albumin: reduces zeta potential, bringing the RBCs closer together and enhancing agglutination. Albumin does not contribute much to antibody uptake. Longer incubation time is needed with this media than with the previously discussed media. Detection of some IgG antibodies can be enhanced with enzyme test methods. Proteolytic enzymes (papain and ficin) denature some RBC antigens and remove negative charges from the RBC membranes. This reduces the zeta potential, bringing the cells closer together. Enzyme techniques are particularly useful in the identification of Rh antibodies and antibodies in the Kidd, Lewis, P and I systems. However, enzymes destroy some antigens including Fya, Fyb, M, and N. The effect of proteolytic enzymes on the S and s antigens are variable.

Beta Thalassemia MinimaBeta Thalassemia MinorBeta Thalassemia Intermedia Beta Thalassemia MajorDelta-Beta ThalassemiaAnemiaAbsentMild to absentModerateSevereMildRed blood cell (RBC) countNormalIncreasedDecreased to normalDecreasedDecreased to normalHemoglobin(Hb)NormalDecreased to normal (10 - 12 g/dL) Decreased (7 - 10 g/dL)Marked decrease (<7 g/dL)Decreased to normal (8 - 13 g/dL)Mean corpuscular volume (MCV)Slight to no decreaseMarked decreaseMarked decreaseMarked decreaseMay be slightly decreasedMean corpuscular hemoglobin concentration (MCHC)Slight to no decreaseMarked decrease Marked decreaseMarked decreaseMay be slightly decreasedRed blood cell distribution width (RDW)NormalNormal to slightly increasedIncreasedIncreasedNormalRBC morphologyNormalMarked hypochromia and microcytosis Codocytes (target cells) Possible basophilic stippling Nucleated RBCs are usually not presentMarked hypochromia and microcytosis Codocytes (target cells) Possible basophilic stippling Nucleated RBCs are usually not presentMarked hypochromia and microcytosis Codocytes (target cells) schistocytes ovalocytes basophilic stippling polychromasia nucleated RBCs Possible hypochromia and microcytosis Codocytes (target cells) Basophilic stippling Reticulocyte countNormalMay be slightly increasedSlightly increased (<5%)Mildly increased (5 - 10%)Mildly increasedHb electrophoresisNormal patternDecreased amount of Hb A Variable amounts of Hb A2 and Hb F Decreased amount of Hb A Variable amount of Hb A2 Hb F is usually increased Severly decreased amount of Hb A Variable amount of Hb A2 Usually an increased amount of Hb F Decreased amount of Hb A and Hb A2 Increased amount of Hb F (15 - 20%)If red blood cells are normochromic and normocytic, the RBC, Hb, and Hematocrit (HCT) test values follow in three-fold progression (i.e., RBC x 3 = Hb and Hb x 3 = HCT). This is sometimes referred to as "the rule of threes." This rule will usually not apply in cases of beta thalassemia, particularly beta thalassemia minor where the RBCs are not normochromic and are microcytic, and where there is a disproportionate number of RBCs for the amount of hemoglobin that is present.

TestPatient ResultReference Intervals (Adult female)White blood cell (WBC) count 3.7 x 109/L4.4 - 11.3 x 109/LRed blood cell (RBC) count5.6 x 1012/L4.1 - 5.1 x 1012/LHemoglobin (Hb)10.5 g/dL12.3 - 15.3 g/dLHematocrit (HCT)36.6%35.9 - 44.6%MCV65.8 fL80.0 - 96.0 fLMCH19.9 pg27.5 - 33.2 pgMCHC26.7%33.4 - 35.5%RDW14.0<14.5Platelets249.0 x 109/L100.0 - 450.0 x 109/LTotal serum iron165 µg/dL60 - 150 µg/dL Iron-binding capacity230 µg/dL250 - 400 µg/dLThe RBC count is increased for the amount of hemoglobin present. The concentration of hemoglobin in the RBCs is slightly decreased (hypochromic) and the cells are small (microcytic). The variation in RBC size (RDW) is within normal limits.

Up to 5 WBCs per microliter are present in normal adult CSF. Children have slightly higher counts, while in newborns a count of up to 30 leukocytes per microliter is within normal limits. CSF containing up to 200 WBCs or 400 RBCs per microliter may appear clear or only slightly hazy, so all specimens must be examined microscopically.

The following table lists the properties of normal CSF in adults and children: Condition Appearance Predominant Cell normal adult clear, colorless lymph 60% monocytes 30% neutrophil 2% 0-5 WBC / ul 0 RBC / ul normal neonate clear, colorless lymph 20% monocytes 70% neutrophil 4% 0-30 WBC / ul variable RBC Adapted from Saunders Manual of Clinical Laboratory Science. Craig A. Lehrmann, Ed. WB Saunders, 1998.

Three separate loci (ABO, Hh, and Se) contain the genes that control the location and occurrence of the A and B antigens. Hh and Se genes are closely linked on chromosome 19. The precursor substance is acted upon by the H gene and is converted to H substance. The product of the H gene is an enzyme fucosyltransferase, responsible for attaching fucose to the terminal galactose of the precursor substance on the RBC membrane and thus forming H substance. There are only two recognized alleles at this locus: the active form, H, and an amorph, h. The H gene is a high-incidence gene. People who inherit hh are extremely rare. Since the h gene is amorphic, it does not act on the precursor substance.

Erythrocytes are produced in the bone marrow and released into the peripheral blood where they may remain for approximately 120 days before senescence.Their main function is the transport of the respiratory gases (oxygen and carbon dioxide) between the lungs and body tissues.Each erythrocyte can be thought of as an "envelope" containing hemoglobin.Each hemoglobin molecule contains iron which has a high affinity for oxygen.As a result, when an erythrocyte passes through one of the capillaries of the lungs, it picks up oxygen.The oxygen is transported through the blood to the tissues where it is released.Carbon dioxide from the tissues then diffuses into the RBC where it undergoes chemical changes.About 70% of the altered carbon dioxide diffuses into the plasma, 25% binds to the hemoglobin molecule, and 5% goes into simple solution within the red cell.In each of these three ways carbon dioxide is transported from the body tissues back to the lungs, where it is released.

The first group is composed of erythrocytes or red blood cells (RBC's). The main function of the erythrocytes is the transport of oxygen from the lungs to the body tissues. Most of the cells in this Wright's stained peripheral blood smear are red cells. On is shown at the arrowhead.

The nucleus is slightly larger than a normal RBC. It is usually round or oval in shape, but may be slightly indented. The chromatin is very dense and clumped.

Hemolysis can easily be caused by improper phlebotomy techniques. Hemolysis occurs when RBCs are broken up and hemoglobin is released into the plasma, causing it to become pink rather than its natural straw color. Hemolysis can occur by using too small a needle, pulling a syringe plunger too rapidly, expelling blood vigorously into a tube, or shaking a tube of blood too hard. Hemolysis can cause falsely increased potassium, magnesium, iron, and ammonia levels, and other aberrant lab results.In this case, Marcie did not properly wipe the site with gauze after cleaning it with alcohol, and alcohol contacting the blood could have caused RBCs to break up or hemolyze. Marcie also squeezed the baby's foot too hard, causing hemolysis.Relevant topics:Site selection and preparation, Heelstick: Puncture, Hemolysis, Causes of hemolysis

Type of Cell Size Shape Stain Epithelial cells 25 microns irregular pink/red White blood cells 12 microns round pink/red Red blood cells 7 microns round pink/red Yeast 7 microns ovoid blue Bacteria 0.5 - 1 micron variable blue or red Epithelial cells are larger than white blood cells and red blood cells, and contain a single nucleus. White blood cells (neutrophils contained in pus) usually show a segmented nucleus. Red blood cells are 1/2 to 2/3 as large as white blood cells, contain no nucleus, and are Gram negative. Hyphae are gram positive tubular filamentous fungal elements which may show branching or intertwining. Yeast cells are round to oval, often budding, Gram positive fungal elements, about the same size as RBCs. They are generally much larger than bacteria.

Another example of "hyperchromic" cells seen at the edge of a smear. If MCHC is above 36 gms/dl of RBC, recheck hemoglobin and hematocrit; technical error is most likely the cause.

Preanalytical Error What is it? How does it happen? What is the result? Hemolysis Red blood cells (RBCs) break and release contents of cell into plasma. Needle incorrectly positioned in vein; cells forced to squeeze through opening. Needle gauge too small; slow blood return into tube. Vigorous mixing or shaking of tube. Alcohol on skin that has not had sufficient time to dry. Some test results may be falsely elevated. (Potassium is especially affected by hemolysis.) Patient may have to be re-drawn. Clotted specimen Clumped or clotted cells in specimen that requires anticoagulated or whole blood Insufficient mixing of blood with anticoagulant in tube. Delay in mixing tube. Slow filling tube. Inaccurate test results for cell counts and clotting studies. Patient may have to be re-drawn. Tube filled to incorrect volume Too little or too much blood in tube. Tube removed from needle too quickly. Vacuum in tube has been compromised due to use of tube past the expiration date (Results in a short fill). Manual fill of tube may lead to over-fill. Test results may be unreliable due to dilution errors. Patient may have to be re-drawn.

Life-Threatening HemorrhageDespite potential risk, sometimes immediate transfusion is necessary, even for patients with red cell antibodies. In such cases transfusion service staff should alert the medical director, who can discuss options with clinical staff.The medical director will generally talk to the staff attending the patient and indicate that, if possible, they should hold off transfusion. But if it is a case of massive bleeding where exsanguinating hemorrhage is likely, it is better to give some blood and monitor for a delayed hemolytic transfusion reaction than to let the patient bleed to death.Transfusing when bleeding is brisk will result in much of the autologous and incompatible blood bleeding out, with the possibility of a delayed hemolytic reaction once the patient's antibody rebounds and destroys still present antigen-positive donor red cells.Some transfusion services also try to minimize the risk of unmatched blood by typing their emergency supply of O Rh negative RBCs for the K antigen, since anti-K is a relatively common clinically significant antibody. See Resources for two papers that discuss the risks of transfusing un-crossmatched emergency blood.

There are many red and white cells in this high power field. There are a group of five red cells that can be identified on the lower right-hand side of the field. It is important to use the fine focus adjustment on the microscope when trying to differentiate RBCs from WBCs.Supravital stain can help to distinguish RBCs from WBCs. Two RBCs are indicated by blue arrows and a WBC is indicated by the red arrow in the lower image of a supravital-stained sediment/

Yeast can appear as single cells or in the budding form. As single cells they can be confused with RBCs because they are about the same size. In the budding form, yeast is easily identified as demonstrated on this slide. Yeast can be found in patients with cystitis due to yeast, usually candida, or as a vaginal contaminant from patient's with vaginal candidiasis.

The results of this specimen are abnormal but the abnormalities correlate with each other. The turbidity can be explained by the presence of bacteria and crystals. The presence of RBCs in the microscopic explains the blood found on the dipstick. The casts, bacteria and WBCs can account for the increased protein. The results may be reported.

Next the number of RBCs, WBCs, epithelial cells, parasites, and fat will be counted.Move to the center of the coverslip and examine 10 fields under high power (40X objective) brightfield. Use phase-contrast as needed. Determine the average number of each element found and record the findings as number per high power field (HPF). An abundance of any one element may be recorded as >100/HPF when 1/4 field is counted and the total field is estimated to be greater than 200/HPF.

An 80-year-old man was seen in the emergency room with sudden onset of right-side chest pain accentuated on inspiration. His cough was productive of yellow sputum, and he was short of breath. His temperature was 101.2°F. A chest X-ray revealed right middle lobe pneumonia. A complete blood count (CBC) was ordered. The results were as follows:CBC ParameterPatient ResultReference IntervalWBC33.0 x 109/L4.0 - 11.0 x 109/LRBC4.5 x 1012/L4.5 - 5.9 x 1012/LHemoglobin15.2 g/dL13.5 - 17.5 g/dLHematocrit44%41 - 53%Platelet200 x 109/L150 - 450 x 109/LSegmented neutrophil6540 - 80%Band neutrophil100 - 5%Lymphocyte 525 - 35%Eosinophil 30 - 5%Basophil 20 - 2%Monocyte252 - 10%A peripheral smear was reviewed based on the elevated WBC and increased monocyte count. A representative field from the Wright-Giemsa stained smear (1000X magnification) is shown on the right. The cells indicated by the blue arrows are atypical monocytes. They have abundant cytoplasm that is more blue than the typical gray-blue cytoplasm of normal monoctes. A few scattered vacuoles are also present. The atypical monocytes, in company with toxic neutrophils (indicated by the red arrow), appeared to be a response to infection. The patient had a past history of tuberculosis, which may account for the monocytosis.